Galenical formulations

ABSTRACT

Abstract This invention describes galenical formulations that contain perfluoroalkyl-containing dye molecules and other perfluoroalkyl-containing substances. The new formulations are suitable as, i.a., contrast media for near-infrared diagnosis.

[0001] The invention relates to the field of galenical formulations,which are used in particular as contrast media for the visualization oflymph nodes. The invention relates to the subject that is characterizedin the claims, namely new galenical formulations that containperfluoroalkyl-containing dye molecules and otherperfluoroalkyl-containing substances.

[0002] Malignant tumors metastasize heaped in regional lymph nodes,whereby several lymph node stations can also be involved. Thus, lymphnode metastases are found in about 50-69% of all patients with malignanttumors (Elke, Lymphographie [Lymphography], in: Frommhold, Stender,Thurn (eds.), Radiologische Diagnostik in Klinik und Praxis[Radiological Diagnosis in Clinical Studies and Practice], Volume IV,Thieme Verlag Stuttgart, 7th Ed., 434-496, 1984). The diagnosis of ametastatic attack of lymph nodes is of great importance with respect tothe therapy and prognosis of malignant diseases. With the modern imagingmethods (CT, US, and MRT), lymphogenous metastasis sites of malignanttumors are detected only inadequately, since in most cases, only thesize and the shape of the lymph node can be used as a diagnosticcriterion. Thus, small metastases in non-enlarged lymph nodes (<2 cm)are not distinguished from lymph node hyperplasias without a malignantattack (Steinkamp et al., Sonographie und Kernspintomographie:Differentialdiagnostik von reaktiver Lymphknotenvergröberung undLymphknotenmetastasen am Hals [Sonography and Nuclear Spin Tomography:Differential Diagnosis of Reactive Lymph Node Enlargement and Lymph NodeMetastases on the Neck], Radiol. Diagn. 33: 158, 1992).

[0003] It would be desirable to distinguish between lymph nodes withmetastatic attack and hyperplastic lymph nodes with the aid of specificcontrast media. In this case, the contrast medium could be adminisiteredintravasally or interstitially/intracutaneously (see above Siefert, H.M. et al., Lymphology 13, 150-157, 1980). Theinterstitial/intracutaneous administration has the advantage that thesubstance is transported directly from the scattering focus (e.g.,primary tumor) by the corresponding lymph tract into the potentiallyrelated regional lymph node stations. Likewise, a high concentration ofthe contrast medium in the lymph nodes can be achieved with a low dose.Such markers that are to be administered interstitially were mainly usedin the nuclear-medicine evaluation (with use of radioactive particles,such as, e.g., ¹⁹⁸Au-colloid). Nuclear-medicine methods have only a veryinadequate spatial resolution, however, in contrast to nuclear spintomography with its high spatial resolution in the range of fractions ofa millimeter. The direct x-ray-lymphography (injection of an oilycontrast medium suspension in a prepared lymph vessel) is an invasivemethod that is used very rarely and that can visualize only a few lymphoutflow stations. Fluorescence-labeled dextrans are also usedexperimentally in animal experiments to be able to observe the lymphoutflow after their interstitial administration. All commonly usedmarkers for the visualization of lymph tracts and lymph nodes afterinterstitial/intracutaneous administration have in common the fact thatthey are substances with a particulate nature (“particulates,” e.g.,emulsions and nanocrystal suspensions) or large polymers (see above, WO90/14846). The previously described preparations have proven to be ofvalue, however, based on their inadequate local and systemiccompatibility as well as their small lymph passageway, which produces aninadequate diagnostic efficiency, in most cases unsuitable for indirectlymphography.

[0004] There is generally a greet need, therefore, for lymph-specificcontrast-media with suitable pharmaceutical and pharmacologicalproperties. In the pharmaceutical properties, focus is placed first onthe highest possible contrast medium concentration and an adequatestability. In the case of the pharmacological properties, and inaddition to a diagnostically relevant lymph concentration that is asuniform as possible over several (or in the case of intravenousadministration over all) lymph stations, focus is placed mainly on aquick and complete excretion of the contrast medium to avoid anunnecessary load of the entire organism. Moreover, correspondingpreparations must have at their disposal an adequate local and acutecompatibility.

[0005] With respect to the application in radiological practice and inaddition to as simple an application as possible of correspondingpreparations, the quick “start-up” of the preparations is of centralimportance. Thus, if at all possible, it should be possible to performimaging within a few hours after the administration of the contrastmedia.

[0006] Contrast media that are suitable for the visualization of lymphnodes in nuclear spin tomography are described in German Laid-OpenSpecification DE 196 03 033. There, perfluoroalkyl-containing metalcomplexes are disclosed, which are preferably used as lymphographicagents (see FIG. 1 of DE 196 03 033). Similar metal complexes that aresuitable especially as blood-pool agents are described in GermanLaid-Open Specification DE 197 29 013.

[0007] A process for in-vivo diagnosis with use of NIR radiation isdescribed in International Application WO 96/17628. Such diagnosticprocesses are at present under development. The contrast media that aredescribed in this document are not suitable for visualizing the lymphnodes. There is therefore a need for suitable lymph-specific contrastmedia for these new diagnostic processes.

[0008] The object of this invention is therefore to make available newgalenical formulations that are suitable as contrast media especiallyfor the visualization of lymph nodes in the above-mentioned newdiagnostic processes, and that-meet the above-mentioned pharmaceuticaland pharmacological requirements.

[0009] This object is achieved by the galenical formulations of thisinvention.

[0010] The new galenical formulations contain perfluoroalkyl-containingdye molecules, which can be used as contrast media in near-infrareddiagnosis. The dyes satisfy certain photophysical and chemicalrequirements. They have high absorption coefficients and highfluorescence quantum yields to produce an effective signal even at thelowest tissue concentrations. The absorption maxima overlap a widespectral range in a freely selectable manner. For detection in lowertissue layers (several centimeters under the surface), the spectralrange between 600 and 900 nm is essential, while absorption wavelengthsof 400 to 600 nm are sufficient for surface detection. The dyes furtherhave a high chemical stability and photostability. When using light forfluorescence stimulation, the essential problem is the limitedpenetration depth of the light, which lies in the submillimeter range inthe VIS but can be in the centimeter range in the NIR. With respect tothe penetration depth, detection processes in surface tissue diseases,as well as soft tissues, are unproblematic. Since a considerable numberof tissue changes (e.g., breast tumors, skin tumors, lymph node changes)are located on the surface, optical diagnostic processes in addition tothe conventional methods are offered to perform a tissue differentiationbased on different absorption and fluorescence patterns. In this case,the pronounced diffusion of light, which obtains increasing influencewith growing tissue thickness, reduces both the resolution and thecontrast of an optical image. Dyes that are used as so-called opticalcontrast media and are concentrated in the tissues that are to bedetected can result in principle in an increase of the diagnostic valueof optical detection processes, in which they increase the absorption ofthe tissue and provide an additional measurement signal with thedye-specific fluorescence, which can be detected arbitrarily often infront of a low tissue background with high sensitivity.

[0011] Preferred are dyes from the following classes:

[0012] Polymethine dyes, such as, e.g., cyanine dyes, squarilium dyes,croconium dyes, oxonol dyes, merocyanine dyes, cryptocyanine dyes;

[0013] xanthine dyes, such as, e.g., fluorescein and rhodamine andderivatives thereof;

[0014] heteroaromatic, cationic dyes, such as, e.g., oxazines,phenoxazines, thiazines, phenothiazines.

[0015] The new perfluoroalkyl-containing dye molecules are compounds ofgeneral formula I

R_(f)-L-A  (I)

[0016] in which R_(f) represents a straight-chain or branchedperfluoroalkyl radical with 4 to 30 carbon atoms, L stands for a linker,and A stands for a dye molecule.

[0017] Linker L is a direct bond or a straight-chain or branched carbonchain with up to 20 carbon atoms, which can be substituted with one ormore —OH, —COO, —SO₃ groups and/or optionally is interrupted by one ormore —O—, —S—, —CO—, —CONH—, —NHCO—, —CONR—, —NRCO—, —SO₂—, —NH—, —NRgroups or a piperazine, whereby R stands for a C₁ to C₁₀ alkyl radical,which optionally is substituted with one or more OH groups and/or isinterrupted by one or more oxygen atoms.

[0018] Dye molecule A is a dye from the class of the polymethine dyes,xanthine dyes or the heteroaromatic cationic dyes. Dye molecule A ispreferably a cyanine dye, squarilium dye, croconium dye, oxonol dye,merocyanine dye, cryptocyanine dye, fluorescein dye, rhodamine dye,oxazine dye, phenoxazine dye, thiazine dye or phenothiazine dye.Especially preferably dye molecule A is a molecule according to generalformula II:

[0019] in which

[0020] D stands for a fragment that corresponds to general formulas IIIto VI, whereby the position that is characterized with a star means thelinkage with B:

[0021] and in which B stands for a fragment that corresponds to generalformulas VII to XII:

[0022] whereby R¹ and R² represent a C₁-C₄ sulfoalkyl chain, a saturatedor unsaturated, branched or straight-chain C₁- C₅₀ alkyl chain, wherebythe chain or parts of this chain optionally can form one or morearomatic or saturated, cyclic C₅-C₆ units or bicyclic C₁₀ units, andwhereby the C₁-C₅₀ alkyl chain optionally is interrupted by 0 to 15oxygen atoms and/or by 0 to 3 carbonyl groups and/or is substituted with0 to 5 hydroxy groups,

[0023] R³ stands for a radical —COOE¹, —CONE¹E², —NHCOE¹, —NHCONHE¹,—NE¹E², —OE¹, —OSO₃E¹, —SO₃E¹, —SO₂NHE¹, —E¹,

[0024] whereby E¹ and E², independently of one another, represent ahydrogen atom, a C₁-C₄ sulfoalkyl chain, a saturated or unsaturated,branched or straight-chain C₁-C₅₀ alkyl chain, whereby the chain orparts of this chain optionally can form one or more aromatic orsaturated cyclic C₅-C₆ units or bicyclic C₁₀ units, and whereby theC₁-C₅₀ alkyl chain optionally is interrupted by 0 to 15 oxygen atomsand/or by 0 to 3 carbonyl groups, and/or is substituted with 0 to 5hydroxy groups,

[0025] and whereby R⁴ stands for a hydrogen atom, a fluorine, chlorine,bromine or iodine atom, or a branched or straight-chain C₁-C₁₀ alkylchain,

[0026] b means a number 2 or 3,

[0027] and X and Y, independently of one another, mean O, S, —CH═CH— orC(CH₃)₂.

[0028] Especially preferred perfluoroalkyl-containing dye moleculescontain a perfluoroalkyl radical R_(f) with 6 to 12 carbon atoms, alinker L, which consists of a C₁-C₁₀ alkyl group that contains one ormore oxygen atoms and/or one or more —CONH—, —NHCO—, —CONR—, —NRCOgroups, in which R stands for a C₁-C₅ alkyl radical, which can besubstituted with one or more OH groups and a cyanine dye as a dyemolecule. Among the cyanine dyes, indocarbocyanine dyes,indodicarbocyanine dyes and indotricarbocyanine dyes are preferred.Especially preferred dye molecules are the following compounds:

[0029] in which

[0030] p stands for 1, 2 or 3,

[0031] R¹ and R², independently of one another, stand for a4-sulfobutyl, 3-sulfopropyl, 2-sulfoethyl-, 3-methyl-3-sulfopropyl,methyl, ethyl or propyl radical, and

[0032] R³ stands for hydrogen, for a radical —COOE¹, —CONE¹E², —NHCOE¹,—NHCONHE¹, —NE¹E², —OE¹, —OSO₃E¹, —SO₃E¹, —SO₂NHE¹,

[0033] whereby E¹ and E², independently of one another, stand for ahydrogen atom or for a methyl, ethyl or a C₃-C₆ alkyl radical, which isinterrupted by 0 to 2 oxygen atoms and/or by 0 to 1 carbonyl groupsand/or is substituted with 0 to 5 hydroxy groups or stands for apoly(oxyethylene)glycol radical with 2 to 30 —CH₂CH₂O units.

[0034] The galenical-formulations according to the invention furthercontain other perfluoroalkyl-containing compounds, e.g.,perfluoroalkyl-containing metal complexes. Perfluoroalkyl-containingmetal complexes and their production were already described in theGerman Laid-Open Specifications DE 196 03 033, DE 197 29 013 and WO97/26017. These perfluoroalkyl-containing metal complexes are compoundsof general formula XIV

R_(f)-M  (XIV)

[0035] in which R_(f) represents a straight-chain or branchedperfluoroalkyl radical with 4 to 30 carbon atoms, and M is a moleculeportion that contains 1-6 metal complexes.

[0036] Molecule M stands for, for example, a group L-M¹, whereby Lstands for a linker, and M¹ stands for a metal complex with anopen-chain or cyclic chelator, which contains as central atom an atom ofatomic numbers 21-29, 39, 42, 44 or 57-83. In this case, linker L is adirect bond, a methylene group, an —NHCO group, a group

[0037] whereby p¹ means the numbers 0 to 10, q and u, independently ofone another, mean the numbers 0 or 1, and

[0038] R¹ means a hydrogen atom, a methyl group, a —CH₂—OH group, a—CH₂—CO₂H group or a C₂-C₁₅ chain, which optionally is interrupted by 1to 3 oxygen atoms, 1 to 2>CO groups or an optionally substituted arylgroup and/or is substituted with 1 to 4 hydroxyl groups, 1 to 2 C₁-C₄alkoxy groups, 1 to 2 carboxy groups,

[0039] or a straight-chain, branched, saturated or unsaturated C₂-C₃₀carbon chain, which optionally contains 1 to 10 oxygen atoms, 1 to 3—NR¹ groups, 1 to 2 sulfur atoms, a piperazine, a —CONR¹ group, an—NR¹CO group, an —SO₂ group, an —NR¹—CO₂ group, 1 to 2 CO groups, agroup

[0040] optionally substituted aryls and/or is interrupted by thesegroups and/or is optionally substituted with 1 to 3 —OR¹ groups, 1 to 2oxo groups, 1 to 2 —NH—COR¹ groups, 1 to 2 —CONHR¹ groups, 1 to 2(—CH₂)_(p)—CO₂H groups, 1 to 2 groups —(CH₂)_(p)—(O)_(q)—CH₂CH₂—R^(F),

[0041] whereby

[0042] R¹, R^(F) and p and q have the above-indicated meanings, and

[0043] T means a C₂-C₁₀ chain, which optionally is interrupted by 1 to 2oxygen atoms or 1 to 2 —NHCO groups.

[0044] In this case, metal complex M″ stands for the following metalcomplexes:

[0045] a complex of general formula XV

[0046] in which R³, Z¹ and Y are independent of one another, and

[0047] R³ has the meaning of R¹ or —(CH₂)_(m)-L-R^(F), whereby m is 0, 1or 2, and L and R^(F) have the above-mentioned meaning,

[0048] Z¹, independently of one another, mean a hydrogen atom or a metalion equivalent of atomic numbers 21-29, 39, 42, 44 or 57-83,

[0049] Y means —OZ¹, or

[0050] whereby Z¹, L, R^(F) and R³ have the above-mentioned meanings,

[0051] a complex of general formula XVI

[0052] in which R³ and Z¹ have the above-mentioned meanings, and R² hasthe meaning of R¹,

[0053] a complex of general formula XVII

[0054] in which Z¹ has the above-mentioned meaning,

[0055] a complex of general formula XVIII

[0056] in which Z¹ has the above-mentioned meaning, and o and q standfor numbers 0 or 1, and yields the sum o+q=1,

[0057] a complex of general formula XIX

[0058] in which Z¹ has the above-mentioned meaning,

[0059] a complex of general formula XX

[0060] in which Z¹ and Y have the above-mentioned meanings,

[0061] a complex of general formula XXI

[0062] in which R³ and Z¹ have the above-mentioned meanings, and R² hasthe above-mentioned meaning of R¹,

[0063] a complex of general formula XXII

[0064] in which R³ and Z¹ have the above-mentioned meanings,

[0065] a complex of general formula XXIII

[0066] in which R³ and Z¹ have the above-mentioned meanings,

[0067] a complex of general formula XXIV

[0068] in which Z¹, p and q have the above-mentioned meaning, and R² hasthe meaning of R¹,

[0069] a complex of general formula XXV

[0070] in which L, R^(F) and Z¹ have the above-mentioned meanings,

[0071] a complex of general formula XXVI

[0072] in which Z¹ has the above-mentioned meaning.

[0073] Such compounds and production thereof are described in GermanLaid-Open Specification DE 196 03 033 A1 and in International PatentApplication WO 97/26017.

[0074] Molecule portion M according to formula XIV can further exhibitthe following structure:

[0075] whereby

[0076] q¹ is a number 0, 1, 2 or 3,

[0077] K stands for a complexing agent or metal complex or salts thereofof organic and/or inorganic bases or amino acids or amino acid amides,

[0078] X is a direct bond for the perfluoroalkyl group, a phenylenegroup or a C₁-C₁₀ alkyl chain, which optionally contains 1-15 oxygenatoms, 1-5 sulfur atoms, 1-10 carbonyl groups, 1-10 —(NR) groups, 1-2NRSO₂ groups, 1-10 CONR groups, 1 piperidine group, 1-3 SO₂ groups, 1-2phenylene groups or optionally is substituted by 1-3 radicals R^(F), inwhich R stands for a hydrogen atom, a phenyl, benzyl or a C₁-C₁₅ alkylgroup, which optionally contains 1-2 NHCO groups, 1-2 CO groups, 1-5oxygen atoms and optionally is substituted by 1-5 hydroxy, 1-5 methoxy,1-3 carboxy, 1-3 R^(F) radicals,

[0079] Y¹ is a direct bond or a chain of general formula XXVII orXXVIII:

[0080] in which

[0081] R^(1a) is a hydrogen atom, a phenyl group, a benzyl group or aC₁-C₇ alkyl group, which optionally is substituted with a carboxy group,a methoxy group or a hydroxy group,

[0082] Z¹ is a direct bond, a polyglycol ether group with up to 5 glycolunits or a molecule portion of general formula XXIX

—CH(R^(2a))—  (XXIX)

[0083] in which R^(2a) is a C₁-C₇ carboxylic acid, a phenyl group, abenzyl group or a —(CH₂)₁₋₅—NH—K group,

[0084] α represents the binding to the nitrogen atom of the skeletonchain, β represents the binding to the complexing agent or metal complexK,

[0085] and in which variables k and m stand for natural numbers between0 and 10, and 1 stands for 0 or 1, and whereby

[0086] G is a CO or SO₂ group.

[0087] Such compounds and the production thereof are described in GermanLaid-Open Specification DE 197 29 013 A1.

[0088] Molecule portion A according to general formula I further canstand for a group L¹-M², in which L¹ stands for a linker and M² standsfor a metal complex. In this case, linker L¹ is a molecule portionaccording to general formula XXX

[0089] in which

[0090] N represents a nitrogen atom,

[0091] A1 means a hydrogen atom, a straight-chain or branched C₁-C₃₀alkyl group, which optionally is interrupted by 1-15 oxygen atoms and/oroptionally is substituted with 1-10 hydroxy groups, 1-2 COOH groups, aphenyl group, a benzyl group and/or 1-5 —OR⁴ groups, with R⁴ in themeaning of a hydrogen atom or a C₁-C₇ alkyl radical, or B1-R^(F),

[0092] B1 means a straight-chain or branched C₁-C₃₀ alkylene group thatoptionally is interrupted by 1-10 oxygen atoms, 1-5 —NH—CO groups, 1-5—CO—NH groups, by a phenylene group (that is optionally substituted by aCOOH group), 1-3 sulfur atoms, 1-2 —N(B2)—SO₀ groups, and/or 1-2—SO₂—N(B2) groups with B2 in the meaning of A1, an NHCO group, a CONHgroup, an N(B2)—SO₂group, or an —SO₂— N(B2) group and/or optionally issubstituted with radical R^(F),

[0093] and in which a represents the binding to metal complex M², and brepresents the binding to perfluoroalkyl group R^(F).

[0094] In this case, metal complex M² stands for a metal complex ofgeneral formula XXXI

[0095] whereby R¹ stands for a hydrogen atom or a metal ion equivalentof atomic numbers 21-29, 31, 32, 37-39, 42-44, 49 or 57-83,

[0096] R² and R³ stand for a hydrogen atom, a C₁-C₇ alkyl group, abenzyl group, a phenyl group, —CH₂OH or —CH ₂—OCH₃,

[0097] U stands for radical L, whereby L and U, independently of oneanother, can be the same or different, however.

[0098] Such compounds and their production are described in the Germanpatent application with file number 199 14 101.0 as well as in theexamples below.

[0099] Especially preferred are metal complexes in which the centralatom is a gadolinium atom (atomic number 64). Metal complexes withcyclic chelating agents are preferred compared to those with open-chainchelating agents.

[0100] Especially preferred gadolinium complexes are the gadoliniumcomplex of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane(for production, see WO 97/26017, Example 33),

[0101] the gadolinium complex of10-[2-hydroxy-4-aza-5-oxo-7-oxa-10,10,11,11,12,12,13,13,14,14,15,15,16,16,17,17,17-heptadecafluoroheptadecyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane(for production, see DE 196 03 033, Example 2),

[0102]1,4,7-tris{1,4,7-tris(N-carboxylatomethyl)-10-(N-1-methyl-3,6-diaza-2,5,8-trioxooctane-1,8-diyl)-1,4,7,10-tetraazacyclododecane,Gd-complex}-10-(N-2H,2H,4H,4H,5H,5H-3-oxa-perfluoro-tridecanoyl)-1,4,7,10-tetraazacyclododecane(for production, see DE 197 29 013, Example 1),

[0103]1,4,7-tris{1,4,7-tris[(N-carboxylatomethyl)]-10-[N-1-methyl-3-aza-2,5-dioxopentam-1,5-diyl]-1,4,7,10-tetraazacyclododecane,Gdcomplex}-10-[2-(N-ethyl-N-perfluorooctylsulfonyl)-amino]-acetyl-1,4,7,10-tetraazacyclododecane(for production, see DE 197 29 013, Example 12),

[0104] the gadolinium complex of10-[2-hydroxy-4-aza-5-oxo-7-aza-7(perfluorooctylsulfonyl)-nonyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane(for production, see DE 196 03 033, Example 1),

[0105]1,4,7-tris(carboxylatomethyl)-10-[(3-aza-4-oxo-hexan-5-ylic)-acid-N-(2,3-dihydroxy-propyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa)-perfluorotridecyl)-amide}-1,4,7,10-tetraazacyclododecane,gadolinium complex (for production see examples),

[0106]1,4,7-tris(carboxylatomethyl)-10-[(3-aza-4-oxo-hexan-5-ylic)-acid-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa-perfluorotridecyl)-amide]-1,4,7,10-tetraazacyclododecane,gadolinium complex (for production see examples),

[0107]1,4,7-tris(carboxylatomethyl)-10-{(3-aza-4-oxo-hexan-5-ylic)-acid-[N-3,6,9,12,15-pentaoxa)-hexadexyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa)-perfluorotridecyl]-amide]-1,4,7,10-tetraazacyclododecane,gadolinium complex (for production, see examples),

[0108] and1,4,7-tris(carboxylatomethyl)-10-[(3-aza-4-oxo-hexan-5-ylic)-acid-N-(5-hydroxy-3-oxa-pentyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa)-perfluorotridecyl)-amide]-1,4,7,10-tetraazacyclododecane,gadolinium complex (for production, see examples).

[0109] Instead of the perfluoroalkyl-containing metal complexes, otherperfluoroalkyl-containing compounds can also be contained in thegalenical formulations according to the invention. Such compounds arecompounds of general formula XXXII

R_(f)-L²-G¹  (XXXII)

[0110] in which R_(f) represents a straight-chain or branchedperfluoroalkyl radical with 4 to 30 carbon atoms, L² stands for alinker, and G¹ stands for a hydrophilic group.

[0111] Linker L² is a direct bond, an —SO₂ group or a straight-chain orbranched carbon chain with up to 20 carbon atoms, which can besubstituted with one or more —OH, —COO⁻, —SO₃ groups and/or optionallycontains one or more —O—, —S—, —CO—, —CONH—, —NHCO—, —CONR″—, —NR″CO—,—SO₂—, —PO₄—, —NH—, —NR″ groups, an aryl ring or a piperazine, wherebyR″ stands for a C₁ to C₂₀ alkyl radical, which in turn can contain oneor more O-atoms and/or can be substituted with —COO⁻ or SO₃ groups.Hydrophilic group G¹ stands for a monosaccharide or disaccharide, one ormore adjacent —COO⁻ or —SO₃ groups, a dicarboxylic acid, an isophthalicacid, a picolinic acid, a benzenesulfonic acid, a tetrahydropyranedicarboxylic acid, a 2,6-pyridinedicarboxylic acid, a quaternaryammonium ion, an aminopolycarboxylic acid, anaminodipolyethyleneglycolsulfonic acid, an aminopolyethylene glycolgroup, an SO₂—(CH₂)₂—OH group, a polyhydroxyalkyl chain with at leasttwo hydroxyl groups or one or more polyethylene glycol chains with atleast two glycol units, whereby the polyethylene glycol chains areterminated by an —OH or —OCH₃ group. Such substances are already known(see, e.g., Tetrahedron Letters, Vol. 36, No. 4, pp. 539-542, 1995). Thesynthesis of some of these compounds is described in detail in theexamples below. Those compounds that contain a monosaccharide ashydrophilic group G¹ are preferably used.

[0112] Especially preferred perfluoroalkyl-containing compounds containa perfluoroalkyl radical R_(f) with 6 to 12 carbon atoms, a linker L₂,which represents an —SO₂ group or a straight-chain or branched carbonchain with up to 20 carbon atoms, which in turn contains one or more—O—, —CO—, —CONH—, —NHCO—, —CONR″—, NR″CO—, —SO₂ groups or a piperazine,in which R″ has the above-indicated meaning, and a monosaccharide ashydrophilic group G¹.

[0113] It is also possible to produce galenical formulations with threecomponents and to use the latter as contrast media for the visualizationof lymph nodes. Such formulations are described in detail in theexamples below.

[0114] The substance mixtures according to the invention can be presentin dissolved form in a solvent. The solvent is preferably water. Theproportion of the perfluoroalkyl-containing dye molecule is between 0.1and 10 mol % relative to the total amount of perfluoroalkyl-containingsubstances, preferably between 1 and 10 mol %. Preferred are mixturesthat consist of perfluoroalkyl-containing dye molecules and otherperfluroalkyl-containing compounds, in which-the perfluoroalkyl chainshave a length of 6 to 12 carbon atoms. Especially preferred are mixturesin which both the perfluoroalkyl-containing dye molecules and the otherperfluoroalkyl-containing compounds have a perfluoroalkyl chain with 8carbon atoms.

[0115] The new galenical formulations show surprising advantages intheir use as contrast media. Galenical formulations that consist ofperfluoroalkyl-containing dye molecules and otherperfluoroalkyl-containing substances can be produced in a wide variety.These formulations are suitable for fluorescence detection and visuallydetectable staining of lymph nodes after interstitial or intravenousadministration.

[0116] Compared to the already known contrast media for visualization ofthe lymph nodes, they show an improved compatibility and an almostcomplete excretion. The local compatibility further is also higher thanin the previously known contrast media, and the new formulationssimultaneously show a higher organ specificity. The concentration in thelymph nodes is higher than in the known contrast media for lymphography.If perfluoroalkyl-containing dye molecules and perfluorine-containingmetal complexes are used simultaneously, it is possible to use variousdiagnostic processes in succession. In addition to the near-infrareddiagnosis, e.g., computer or nuclear spin tomography can also beperformed.

[0117] Another advantage consists in the fact that the lymph nodesassume a characteristic coloring. In addition to the NIR diagnosis, thisallows an intraoperative fluorescence diagnosis of the lymph nodemorphology and lymph tract permeability as well as afluorescence-supported removal of biopsies. In this case, thefluorescence-supported detection of the so-called sentinel lymph node isespecially important. The sentinel lymph node is the first lymph nodethat drains the lymphs of a tumor area and thus also the first lymphnode that is affected in a metastasis attack of lymph nodes.

[0118] The fluorescence-supported detection of these lymph nodes iscarried out very much more simply than, e.g., the detection with the aidof radiopharmaceutical agents, since the detection of x-ray radiation isalways more difficult than direct detection of fluorescence radiation.In addition, the stained lymph nodes are to be made visible, since thelymph nodes also can assume a characteristic coloring in the case of thecorresponding dosage of the compounds according to the invention.

[0119] The dye molecules are produced in a way that is similar tomethods that are known in the literature and then coupled withperfluoroalkyl derivatives. Preferred are dyes from the above-mentionedclasses, which contain carboxyl groups or isothiocyanate groups.Especially preferred are those dyes that contain carboxyl groups, whichafter activation with use of standard reagents are reacted with aminogroups that contain perfluoroalkyl derivatives with the formation of anamide group. Literature for synthesis of polymethine dyes: BioconjugateChem. 4, 105-111, 1993; Bioconjugate Chem. 7, 356-62, 1996; BioconjugateChem. 8, 751-56, 1997; Cytometry 10, 11-19, 1989 and 11, 418-30, 1990;J. Heterocycl. Chem. 33, 1871-6, 1996; J. Org. Chem. 60, 2391-5, 1995;Dyes and Pigments 17, 19-27, 1991, Dyes and Pigments 21, 227-34, 1993;J. Fluoresc. 3, 153-155, 1993; Anal. Biochem. 217, 197-204, 1994; U.S.Pat. No. 4,981,977; U.S. Pat. No. 5,688,966; U.S. Pat. No. 5,808,044; WO97/42976; WO 97/42978; WO 98/22146; WO 98/26077; EP 0800831.

[0120] The production of the galenical formulations is carried out inthat the perfluoroalkyl-containing dye molecule (component A) and theother perfluoroalkyl-containing substance (component B) are weighed andare dissolved in a suitable solvent. An especially suitable solvent iswater. As already mentioned above, the proportion of theperfluoroalkyl-containing dye molecule is between 1 and 10 mol %relative to the total amount of perfluoroalkyl-containing substances.The concentration of the solution is preferably between 0.1 mmol/L and20 mmol/L relative to the dye. This solution is then added in excess tocommonly used galenical additives, such as, e.g., buffer solutions andthe Ca-salt of the complexing agent. At 10 to 100° C., the solutions arestirred vigorously. As an alternative, the solutions can be treated at10 to 100° C. in an ultrasound bath. Another alternative consists inthat the solutions are treated with microwaves.

[0121] In substances that do not dissolve in water as individualcomponents, it has proven advantageous to add a solubilizer, such asalcohol (e.g., methanol or ethanol) or another water-miscible solventand then to distill off the latter slowly. The distillation can takeplace under vacuum. The residue is then dissolved in water, and thesolution is filtered. It is also possible to dissolve each component perse separately in a solvent, then to join them and to proceed further asabove.

[0122] Such produced solutions can be freeze-dried. The freeze-driedsolutions can be dissolved again in water and retain, surprisinglyenough, their advantageous properties. This allows a long storage timeof the active ingredient.

[0123] With use as a contrast medium for the visualization of the lymphnodes, the aqueous solutions of the perfluoroalkyl-containing substances(at a concentration of between 0.1 mmol/L and 20 mmol/L relative to thedye, see above) are administered by interstitial/intracutaneousinjection or intravenous injection at one or more injection sites. Theadministration volume relates to the species and form of administrationbetween 0.1 ml and 30 ml, and the administered dose is preferablybetween 0.1 μmol/kg and 10 μmol/kg of body weight, relative to the dye.

[0124] After the galenic formulation is injected, light from thecorresponding spectral range is irradiated for electronic stimulation ofthe dye that is used in the tissue. The reflected stimulation light orthe fluorescence radiation that is emitted by the dye is recorded.Preferred are the methods in which the tissue irradiates over a largesurface and the fluorescence radiation is triggered locally by recordingwith a CCD camera or the tissue areas that are to be formed are rasteredwith a fiber optic light guide, and the signals that are obtained areassembled by computer into a synthetic image. In this case, fluorescencecan be detected and evaluated spectrally and/or by phase selection, aswell as in a steady-state manner and/or in a time-resolved manner. Thefluorescence images that are obtained can be produced at the same timeas the white light images and are depicted above one another in a figurefor data evaluation. In the case of intraoperative diagnosis, thestaining can be observed visually in addition for fluorescencedetection.

[0125] The following examples explain the invention, without intendingthat it be limited to these examples.

EXAMPLES 1 AND 2 Synthesis of bis-sulfobutyl-indocyanine dyes1,1′-bis-(4-sulfobutyl)-indodicarbocyanine-5-carboxylic acid, sodiumsalt (1) and 1,1′-bis-(4-sulfobutyl)indotricarbocyanine-5-carboxylicacid, sodium salt (2) for coupling to perfluoroalkylamino derivatives

[0126] Synthesis is generally carried out starting from1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine and1-(4-sulfobutyl)-2,3,3-trimethyl-5-carboxy-3H-indolenine (Cytometry 10,11-19, 1989, Talanta 39, 505-510, 1992).

EXAMPLE 1

[0127] Synthesis of1,1′-bis-(4-sulfobutyl)indodicarbocyanine-5-carboxylic acid. sodium salt(1)

[0128] 1.2 g (4.1 mmol) of1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine and 1.0 g (3.9 mmol) ofmalonaldehyde-bis-phenylimine-hydrochloride are stirred into 15 ml ofacetic acid anhydride for 30 minutes at 120° C. and then cooled to roomtemperature with a water bath. Then, 1.4 g (4.2 mmol) of1-(4-sulfobutyl)-2,3,3-trimethyl-5-carboxy-3H-indolenine, 1.2 g (14.6mmol) of anhydrous sodium acetate, 15 ml of acetic acid anhydride and 6ml of acetic acid are added in succession. The reaction mixture isheated for 1 hour to 120° C., the reaction solution is cooled and mixedwith 100 ml of ether. The precipitated solid is filtered off.Chromatographic purification is carried out on RP-silica gel EUROPREP60-30 C18 (Knauer), 60A, 20-45μ (eluant: water/MeOH, step gradient of 0%to 70% MeOH). Methanol is removed from the product-containing fractionsin a rotary evaporator, and the fractions are then freeze-dried, yield:1.8 g (66%), blue lyophilizate.

EXAMPLE 2

[0129] Synthesis of1,1′-bis-(4-sulfobutyl)indotricarbocyanine-5-carboxylic acid, sodiumsalt (2)

[0130] 1.2 g (4.1 mmol) of1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine and 1.1 g (3.9 mmol) ofglutaconaldehyde-dianilhydrochloride are stirred in 15 ml of acetic acidanhydride for 30 minutes at 120° C. and then cooled to room temperaturewith a water bath. Then, 1.4 g (4.2 mmol) of1-(4-sulfobutyl)-2,3,3-trimethyl-5-carboxy-3H-indolenine, 1.2 g (14.6mmol) of anhydrous sodium acetate, 15 ml of acetic acid anhydride and 6ml of acetic acid are added. The reaction mixture is heated for 1 hourto 120° C., the now blue solution is cooled and mixed with 100 ml ofether. The working-up and purification are carried out as described inExample 1, yield: 1.8 g (60%) of blue lyophilizate.

EXAMPLES 3 AND 4 Synthesis of mono-sulfobutyl-indocyanine dyes1-methyl-1′-(4-sulfobutyl)indodicarbocyanine-5-carboxylic acid (3) and1-methyl-1′-(4-sulfobutyl)indotricarbocyanine-5-carboxylic acid (4) forcoupling to perfluoroalkylamino derivatives

[0131] The synthesis is generally carried out starting from1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine and1,2,3,3-tetramethyl-5-carboxy-3H-indolium iodide (Cytometry 10, 11-19,1989, Talanta 39, 505-510, 1992).

EXAMPLE 3 Synthesis of1-methyl-1′-(4-sulfobutyl)indodicarbocyanine-5-carboxylic acid (3)

[0132] 1.2 g (4.1 mmol) of1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine and 1.0 g (3.9 mmol) ofmalonaldehyde-bis-phenylimine-hydrochloride are stirred into 15 ml ofacetic acid anhydride for 30 minutes at 120° C. and then cooled to roomtemperature with a water bath. Then, 1.6 g (4.6 mmol) of1,2,3,3-tetramethyl-5-carboxy-3H-indolium iodide, 1.2 g (14.6 mmol) ofanhydrous sodium acetate, 15 ml of acetic acid anhydride and 6 ml ofacetic acid are added in succession. The reaction mixture is heated for1 hour to 120° C., the now blue solution is cooled and mixed with 100 mlof ether. The precipitated solid is filtered off. A chromatographicpurification of RP-silica gel EUROPREP 60-30 C18 (Knauer), 60A, 20-45μis carried out (eluant: water/MeOH, step gradient of 30% to 90% MeOH).Methanol is removed from the product-containing fractions in a rotaryevaporator and then freeze-dried, yield: 0.9 g (42%), blue lyophilizate.

EXAMPLE 4 Synthesis of1-methyl-1′-(4-sulfobutyl)indotricarbocyanine-5-carboxylic acid (4)

[0133] 1.2 g (4.1 mmol) of1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine and 1.1 g (3.9 mmol) ofglutaconaldehyde-dianilhydrochloride are stirred in 15 ml of acetic acidanhydride for 30 minutes at 120° C. and then cooled to room temperaturewith a water bath. Then, 1.6 g (4.6 mmol) of1,2,3,3-tetramethyl-5-carboxy-3H-indolium iodide, 1.2 g (14.6 mmol) ofanhydrous sodium acetate, 15 ml of acetic acid anhydride and 6 ml ofacetic acid are added. The reaction mixture is heated for 1 hour to 120°C., the reaction solution is cooled and mixed with 100 ml of ether. Theworking-up and purification are carried out as described in Example 3.Yield: 1.4 g (62%) of blue lyophilizate.

EXAMPLES 5 TO 8 Synthesis of perfluoroalkylated bis-sulfobutyl-aninedyes 7 to 10

[0134] The synthesis is carried out from 1 to 2 by activation ofcarboxylic acid and reaction with1H,1H,2H,2H,4H,4H,5H,5H-3-oxa-perfluorotridecylamine (5) to 7 and 8, andby reaction withN-(2,3-dihydroxypropyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa-perfluorotridecyl)-amine(6) to 9 and 10. The synthesis of 5 and is described in DE 199 14 101. #M (g/mol) 7 1182.0 5-{N-[2-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyloxy)ethyl]-aminocarbonyl}-2-{7-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-ylidene]-1,3-pentadienyl}-3,3-dimethyl-1-(4- sulfobutyl)-3H-indolium,internal salt, sodium salt 8 1208.05-{N-[2-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro-decyloxy)ethyl]-aminocarbonyl}-2-{7-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-ylidene[-1,3,5-heptatrienyl}-3,3-dimethyl-1- (4-sulfobutyl)-3H-indolium,internal salt, sodium salt 9 1256.15-{N-[2-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyloxy)ethyl]-N-(2,3-dihydroxypropyl)aminocarbonyl}-2-{7-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-ylidene]-1,3-pentadienyl}-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium, internal salt, sodium salt 10 1282.1 5-{N-[2-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyloxy)ethyl]-N-(2,3-dihydroxypropyl)aminocarbonyl}-2-{7-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-ylidene]-1,3,5-heptatrienyl}-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium, internal salt, sodium salt

EXAMPLES 5 AND 6 Synthesis of 7 and 8 from dyes 1 and 2

[0135] A solution of 0.5 mmol of dye 1 or 2 and 0.1 g (1.0 mmol) oftriethylamine in 20 ml of dimethylformamide is mixed at 0° C. with 0.5mmol of TBTU in 10 ml of dimethylformamide and stirred for 15 minutes at0° C. Then, a solution of 0.29 g (0.55 mmol) of 5 and 0.6 mmol oftriethylamine in 5 ml of dimethylformamide is added in drops, and thereaction mixture is stirred for 2 hours at room temperature. After 50 mlof hexane/50 ml of ethyl acetate is added, the precipitated solid isfiltered off and chromatographically purified on RP-silica gelLiChroprep® RP-8 (Merck), 40-63μ (eluant: water/MeOH, step gradient of20% to 80% MeOH); yields: 0.42 g (71%) of 7, 0.45 g (75%) of 8.

EXAMPLES 7 AND 8 Synthesis of 9 and 10 from dyes 1 and 2

[0136] A solution of 0.5 mmol of dye 1 or 2 and 0.1 g (1.0 mmol) oftriethylamine in 20 ml of dimethylformamide is mixed at 0° C. with 0.5mmol of TBTU in 10 ml of dimethylformamide and stirred for 15 minutes at0° C. Then, a solution of 0.37 g (0.65 mmol) of 6 in 5 ml ofdimethylformamide is added in drops, and the reaction mixture is stirredfor 18 hours at room temperature. After 50 ml of hexane/50 ml of ethylacetate is added, the precipitated solid is filtered off and purifiedchromatographically on RP-silica gel LiChroprep® RP-8 (Merck), 40-63μ(eluant: water/MeOH, step gradient of 20% to 80% MeOH); yields: 0.40 g(63%) of 9, 0.43 g (67%) of 10.

EXAMPLES 9 TO 12 Synthesis of perfluoroalkylated mono-sulfobutyl-cyaninedyes 11 to 14

[0137] The synthesis that is embodied in the example is carried out from3 or 4 by activation of the carboxylic acid and reaction with1H,1H,2H,2H,4H,4H,5H,5H-3-oxa-perfluorotridecylamine (5) to 11 and 12,and by reaction withN-(2,3-dihydroxypropyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa-perfluorotridecyl)-amine(6) to 13 and 14. # M (g/mol) 11 1037.92-[5-(1-Ethyl)-1,3-dihydro-3,3-dimethyl-2H-indol-2-ylidene)-1,3-pentatrienyl]-5-{N-[2-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10- heptadecafluorodecyloxy)ethyl]-aminocarbonyl}-3,3-dimethyl-1-(4-sulfobutyl)- 3H-indolium, internalsalt, sodium salt 12 1063.9 2-[5-(1-ethyl)-1,3-dihydro-3,3-dimethyl-2H-indol-2-ylidene)-1,3,5-heptatrienyl]-5-{N-[2-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10- heptadecafluorodecyloxy)-ethyl]-aminocarbonyl}-3,3-dimethyl-1-(4-sulfobutyl)- 3H-indolium, internalsalt, sodium salt 13 1112.9 2-[5-(1-ethyl)-1,3-dihydro-3,3-dimethyl-2H-indol-2-ylidene)-1,3-pentatrienyl]-5-{N-[2-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyloxy)ethyl]-N-(2,3-dihydroxypropyl)aminocarbonyl}-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium, internal salt, sodium salt 14 1138.92-[5-(1-ethyl)-1,3-dihydro-3,3-dimethyl-2H- indol-2-ylidene)-1,35-heptatrienyl]-5-{N-[2- (3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyloxy)-ethyl]-N-(2,3-dihydroxypropyl)aminocarbonyl}-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium, internal salt, sodium salt

EXAMPLES 9 AND 10 Synthesis of 11 and 12 from dyes 3 and 4

[0138] A solution of 0.5 mmol of dye 3 or 4 and 0.1 g (1.0 mmol) oftriethylamine in 20 ml of dimethylformamide is mixed at 0° C. with 0.5mmol of TBTU in 10 ml of dimethylformamide and stirred for 15 minutes at0° C. Then, a solution of 0.29 g (0.55 mmol) of 5 and 0.6 mmol oftriethylamine in 5 ml of dimethylformamide is added in drops, and thereaction mixture is stirred for 2 hours at room temperature. After 100ml of hexane is added, the precipitated solid is filtered off andpurified chromatographically on RP-silica gel LiChroprep® RP-8 (Merck),40-63μ (eluant: water/MeOH, step gradient of 30% to 90% MeOH); yields:0.25 g (48%) of 11, 0.35 g (66%) of 12.

EXAMPLES 11 AND 12 Synthesis of 13 and 14 from dyes 3 and 4

[0139] A solution of 0.5 mmol of dye 1 or 2 and 0.1 g (1.0 mmol) oftriethylamine in 20 ml of dimethylformamide is mixed at 0° C. with 0.5mmol of TBTU in 10 ml of dimethylformamide and stirred for 15 minutes at0° C. Then, a solution of 0.37 g (0.65 mmol) of 6 in 5 ml ofdimethylformamide is added in drops, and the reaction mixture is stirredfor 18 hours at room temperature. After 100 ml of hexane is added, theprecipitated solid is filtered off and purified chromatographically onRP-silica gel LiChroprep® RP-8 (Merck), 40-63μ (eluant: water/MeOH, stepgradient of 20% to 80% MeOH); yields: 0.39 g (70%) of 13, 0.41 g (72%)of 14.

[0140] The following compounds were obtained analogously by reactionwith the described perfluoroalkyl derivatives:

[0141] 5-Carboxyfluorescein-perfluoroalkylamide

[0142] 5-Carboxy-rhodamine6G-perfluoroalkylamide

[0143] 5-Carboxy-X-rhodamine-perfluoroalkylamide

EXAMPLE 13 Production of galenical formulations of gadoliniumcomplex-perfluoroalkyl derivatives in the example of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluoroctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecanewith 1, 2, 5 and 10 mol % of dyes 7 to 14

[0144] 118 mg (100 μmol) of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane(for production, see WO 97/26017, Example 33) is dissolved together with1 μmol, 2 μmol, 5 μmol or 10 μmol of 7 to 14 in 50 ml of water for 20minutes in an ultrasound bath. Then, a dialysis to separate free dyes isperformed (Amicon cell, cut-off 10,000, dialysis volumes 5×100 ml). Theproduct solution is set at a volume of 5 ml (20 mmol/l) of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane)with water.

[0145] In addition to10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,the following Gd-complex perfluoroalkyl derivatives were used for theproduction of the galenical formulations:

[0146] Gadolinium complex of10-[2-hydroxy-4-aza-5-oxo-7-oxa-10,10,11,11,12,12,13,13,14,14,15,15,16,16,17,17,17-heptadecafluoro-heptadecyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane(for production, see DE 196 03 033, Example 2)

[0147] Gadolinium complex of10-[2-hydroxy-4-aza-5-oxo-7-aza-7-(perfluorooctylsulfonyl)-nonyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane(for production, see DE 196 03 033, Example 1)

[0148]1,4,7-Tris(carboxylatomethyl)-10-[(3-aza-4-oxo-hexan-5-ylic)-acid-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa-perfluorotridecyl)-amide]-1,4,7,10-tetraazacyclododecane,gadolinium complex

[0149] Production

[0150] 10 g (15.88 mmol) of the gadolinium complex of10-[1-(carboxymethylcarboamoyl)-ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid and 1.35 g (31.76 mmol) of lithium chloride and 3.66 g (31.76 mmol)of N-hydroxysuccinimide are dissolved at 60° C. in 100 ml of dimethylsulfoxide. It is cooled to 15° C., and 3.51 (17 mmol) ofN,N′-dicyclohexylcarbodiimide is added and stirred for 5 hours at 15° C.To separate the urea, the solution is filtered. 8.63 g (15.88 mmol) of1H,1H,2H,2H,4H,4H,5H,5H-3-oxa-perfluorotridecylamine, hydrochloride and5.06 g (50 mmol) of triethylamine are added to the filtrate and stirredfor 12 hours at room temperature. The solution is poured into 1,500 mlof diethyl ether/100 ml of acetone and stirred for 30 minutes. Theprecipitated solid is filtered off and chromatographed on silica gelRP-18 (mobile solvent: gradient that consists oftetrahydrofuran/acetonitrile/water).

[0151] Yield: 13.86 g (78% of theory) of a colorless, amorphous powder

[0152] Water content: 9.3%

[0153] Elementary analysis (relative to anhydrous substance): Cld: C33.28 H 3.42 N 7.51 F 28.87 Gd 14.05 Fnd: C 33.12 H 3.61 N 7.37 F 28.69Gd 13.89

[0154]1,4,7-Tris(carboxylatomethyl)-10-[(3-aza-4-oxo-hexan-5-ylic)-acid-N-(2,3-dihydroxypropyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa)-perfluorotridecyl)-amide]-1,4,7,10-tetraazacyclododecane,gadolinium complex

[0155] Production

[0156] a) 2H,2H,4H,4H,5H,5H-3-Oxa)-perfluorotridecanoicacid-N-(2,3-dihydroxypropyl)-amide

[0157] 8.90 g (70 mmol) of oxalyl chloride is added to 30 g (57.45 mmol)of 2H,2H,4H,4H,5H,5H-3-oxa-perfluorotridecanoic acid in 300 ml ofdichloromethane, and it is stirred for 12 hours at room temperature. Itis evaporated to the dry state in a vacuum. The residue is dissolved in100 ml of dichloromethane and added in drops at 0° C. to a solution of5.47 g (60 mmol) of 2,3-dihydroxypropylamine and 6.07 g (60 mmol) oftriethylamine, dissolved in 200 ml of dichloromethane. It is stirred for3 hours at 0° C., then for 6 hours at room temperature. 300 ml of 5%aqueous hydrochloric acid is added, and it is thoroughly stirred for 15minutes.- The organic phase is separated, dried on magnesium sulfate andevaporated to the dry state in a vacuum. The residue is chromatographedon silica gel (mobile solvent: dichloromethane/ethanol=15:1).

[0158] Yield: 29.70 g (87% of theory) of a colorless solid

[0159] Elementary analysis: Cld: C 30.32 H 2.20 N 2.36 F 54.35 Fnd: C30.12 H 2.41 N 2.18 F 54.15

[0160] b)N-(2,3-Dihydroxypropyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa-perfluorotridecyl)-amine

[0161] 30 g (48.8 mmol) of the title compound of Example a is dissolvedin 300 ml of tetrahydrofuran, and 50 ml of 10 M borane dimethyl sulfide(in tetrahydrofuran) is added. It is refluxed for 16 hours. It is cooledto 0° C., and 300 ml of methanol is added in drops, then it isevaporated to the dry state in a vacuum. The residue is taken up in amixture of 300 ml of ethanol/50 ml of 10% aqueous hydrochloric acid andstirred for 8 hours at 60° C. It is evaporated to the dry state in avacuum, the residue is taken up in 300 ml of 5% aqueous sodium hydroxidesolution and extracted three times with 300 ml of dichloromethane each.The organic phases are dried on magnesium sulfate, evaporated to the drystate in a vacuum, and the residue is chromatographed on silica gel(mobile solvent: dichloromethane/methanol=15:1).

[0162] Yield: 24.07 g (85% of theory) of a colorless solid

[0163] Elementary-analysis: Cld: C 31.05 H 2.61 N 2.41 F 55.66 Fnd: C31.91 H 2.78 N 2.33 F 55.47

[0164] c)1,4,7-Tris(carboxylatomethyl)-10-[(3-aza-4-oxo-hexan-5-ylic)-acid-N-(2,3-dihydroxypropyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa)-perfluorotridecyl)-amide]-1,4,7,10-tetraazacyclododecane,gadolinium complex

[0165] 10 g (15.88 mmol) of the gadolinium complex of10-[1-(carboxymethylcarboamoyl)-ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid and 1.35 g (31.76 mmol) of lithium chloride are dissolved at 60° C.in 100 ml of dimethyl sulfoxide. It is cooled to 15° C., and 9.21 (15.88mmol) of the title compound of Example b is added. It is stirred for 10minutes, and then 7.42 g (30 mmol) of2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline is added. It is stirredfor 12 hours at room temperature. The solution is poured into a mixtureof 200 ml of acetone/1300 ml of diethyl ether and stirred for 2 hours atroom temperature. The deposited precipitate is filtered off, dissolvedin a mixture that consists of a little ethanol/water and chromatographedon silica gel RP-18 (mobile solvent: gradient that consists oftetrahydrofuran/acetonitrile/water).

[0166] Yield: 16.09 g (85% of theory) of a colorless, amorphous powder

[0167] Water content: 6.3%

[0168] Elementary analysis (relative to anhydrous substance): Cld: C34.26 H 3.64 N 7.05 F 27.10 Gd 13.19 Fnd: C 34.12 H 3.83 N 6.91 F 26.88Gd 12.93

[0169]1,4,7-Tris(carboxylatomethyl)-10-[(3-aza-4-oxo-hexan-5-ylic)-acid-N-(5-hydroxy-3-oxa-pentyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa)-perfluorotridecyl)-amide]-1,4,7,10-tetraazacyclododecane,gadolinium complex

[0170] Production

[0171] a) 2H,2H,4H,4H,5H,5H-3-Oxa-perfluorotridecanoicacid-N-(5-hydroxy-3-oxa-pentyl)-amide

[0172] 8.90 g (70 mmol) of oxalyl chloride is added to 30 g (57.45 mmol)of 2H,2H,4H,4H,5H,5H-3-oxa-perfluorotridecanoic acid in 300 ml ofdichloromethane and stirred for 12 hours at room temperature. It isevaporated to the dry state in a vacuum. The residue is dissolved in 100ml of dichloromethane and added in drops at 0° C. to a solution of 6.25g (60 mmol) of 5-hydroxy-3-oxa-pentylamine and 6.07 g (60 mmol) oftriethylamine, dissolved in 200 ml of dichloromethane. It is stirred for3 hours at 0° C., then for 6 hours at room temperature. 300 ml of 5%aqueous hydrochloric acid is added, and it is thoroughly stirred for 15minutes. The organic phase is separated, dried on magnesium sulfate andevaporated to the dry state in a vacuum. The residue is chromatographedon silica gel (mobile solvent: dichloromethane/acetone=15:1).

[0173] Yield: 32.20 g (92% of theory) of a colorless solid

[0174] Elementary analysis: Cld: C 31.54 H 2.65 N 2.30 F 53.01 Fnd: C31.61 H 2.84 N 2.14 F 52.85

[0175] b)N-(5-Hydroxy-3-oxa-pentyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa-perfluorotridecyl)-amine

[0176] 30 g (49.24 mmol) of the title compound of Example a is dissolvedin 300 ml of tetrahydrofuran, and 31 ml of 10 M borane dimethyl sulfide(in tetrahydrofuran) is added. It is refluxed for 16 hours. It is cooledto 0° C., and 200 ml of methanol is added in drops, then it isevaporated to the dry state in a vacuum. The residue is taken up in amixture of 300 ml of ethanol/50 ml of 10% aqueous hydrochloric acid andstirred for 10 hours at 50° C. It is evaporated to the dry state in avacuum, the residue is taken up in 300 ml of 5% aqueous sodium hydroxidesolution and extracted three times with 300 ml of dichloromethane each.The organic phases are dried on magnesium sulfate, evaporated to the drystate in a vacuum, and the residue is chromatographed on silica gel(mobile solvent: dichloromethane/2-propanol=20:1).

[0177] Yield: 26.09 g (89% of theory) of a colorless solid

[0178] Elementary analysis: Cld: C 32.28 H 3.05 N 2.35 F 54.25 Fnd: C32.12 H 3.21 N 2.18 F 54.09

[0179] c)1,4,7-Tris(carboxylatomethyl)-10-[(3-aza-4-oxo-hexan-5-ylic)-acid-N-(5-hydroxy-3-oxa-pentyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa)-perfluorotridecyl)-amide]-1,4,7,10-tetrazacyclododecane,gadolinium complex

[0180] 10 g (15.88 mmol) of the gadolinium complex of10-[1-(carboxymethylcarboamoyl)-ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid and 1.35 g (31.76 mmol) of lithium chloride are dissolved at 60° C.in 100 ml of dimethyl sulfoxide. It is cooled to 15° C., and 9.45 (15.88mmol) of the title compound of Example b is added. It is stirred for 10minutes, and then 7.42 g (30 mmol) of2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline is added. It is stirredfor 12 hours at room temperature. The solution is poured into a mixtureof 200 ml of acetone/1300 ml of diethyl ether and stirred for 2 hours atroom temperature. The deposited precipitation is filtered off, dissolvedin a mixture that consists of a little ethanol/water and chromatographedon silica gel RP-18 (mobile solvent: gradient that consists oftetrahydrofuran/acetonitrile/water).

[0181] Yield: 16.10 g (84% of theory) of a colorless, amorphous powder

[0182] Water content: 5.7%

[0183] Elementary analysis (relative to anhydrous substance): Cld: C34.83 H 3.84 N 6.96 F 26.76 Cd 13.03 Fnd: C 34.65 H 3.96 N 6.84 F 26.62Cd 12.91

EXAMPLE 14 Production of galenical formulations withmonosaccharide-perfluoroalkyl derivatives in the example of6-[1-O-α-D-mannopyranosyl)-hexanoic acidN-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide and dyes 7 to14 A) Production of 6-[1-O-α-D-mannopyranosyl]-hexanoic acidN-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide

[0184] a) 1,2,3,4,6-Penta-O-acetyl-α,β-D-mannopyranose

[0185] Analogously, as described in the literature [M. L. Wolfrom and A.Thompson in Methods in Carbohydrate Chemistry (R. L. Whistler, M. L.Wolfrom and J. N. BeMiller, Eds.), Academic Press, New York, Vol. II,53, pp. 211-215, (1963)], the reaction of 150 g (832.5 mmol) ofα,β-D-mannopyranose with a mixture that consists of 1,500 ml of absolutepyridine and 1,500 ml of acetic acid anhydride provides, afterworking-up, 315 g (96.7%) of the above-mentioned title compound as acrude product in the form of a viscous and colorless oil. By¹H-NMR-spectroscopic study of the title compound that is thus obtained,the α to β-ratio of both anomers was found to be 4:1. A separation ofthe α,β-anomers of the above-mentioned title compound can be dispensedwith for performing the reaction steps below.

[0186] Elementary analysis: Cld: C 49.21 H 5.68 Fnd: C 49.12 H 5.78

[0187] b) 6-[1-O-α-(2,3,4,6-Tetra-O-acetyl-D-mannopyranosyl)-hexanoicacid ethyl easter]

[0188] Analogously, as described in the literature for the synthesis ofaryl glycopyranosides [J. Conchie and G. A. Levvy in Methods inCarbohydrate Chemistry (R. L. Whistler, M. L. Wolfrom and J. N.BeMiller, Eds.), Academic Press, New York, Vol. II, 90, pp. 345-347,(1963)], the reaction of 156.2 g (400 mmol) of the title compound ofExample Aa) as an α,β-anomer mixture with 67 ml (400 mmol) of6-hydroxy-hexanoic acid ethyl ester and 60.8 ml (520 mmol) of tin(IV)chloride in a total of 600 ml of 1,2-dichloroethane results in theformation of 100.05 g (51% of theory) of the above-mentioned titlecompound as a colorless and viscous oil after column-chromatographicpurification (eluant: hexane/ethyl acetate 2:1). ¹H-NMR-spectroscopicstudy of the title compound that is thus obtained showed that theabove-mentioned title compound is only the pure α-anomer.

[0189] Elementary analysis: Cld: C 52.94 H 6.77 Fnd: C 52.80 H 6.78

[0190] c) 6-[1-O-α-(2,3,4,6-Tetra-O-benzyl-D-mannopyranosyl)-hexanoicacid

[0191] A stirred suspension of 141.0 g (289 mmol) of the title compoundof Example Ab) in 200 ml of dioxane is mixed at room temperature andwith simultaneous vigorous stirring in portions with a total of 238.5 g(4.26 mol) of finely powdered potassium hydroxide powder. To increasethe stirrability, the reaction mixture is mixed with another 200 ml ofdioxane, and the suspension that is thus obtained is subsequently heatedto boiling and mixed drop by drop at this temperature with a total of372 ml (3.128 mol) of benzyl bromide over a period of two hours. After areaction time of 4 hours at 110° C. followed by 12 hours at roomtemperature, the reaction mixture is slowly poured into a total of 2.5liters of ice water for the purpose of working-up, and the water phaseis subsequently completely extracted with diethyl ether. After the etherphase that is thus obtained is washed and the same is subsequently driedon sodium sulfate, salt is suctioned out, and the diethyl ether isremoved in a vacuum. Excess benzyl bromide is then quantitativelydistilled off from the reaction mixture in an oil pump vacuum at an oilbath temperature of 180° C. The resinous-oily residue that is thusobtained is purified on silica gel with use of ethyl acetate/hexane(1:10) as an eluant.

[0192] Yield: 172.2 g (91.0% of theory) of the above-mentioned titlecompound in the form of a colorless and extremely viscous oil

[0193] Elementary analysis: Cld: C 75.68 H 7.16 Fnd: C 75.79 H 7.04

[0194] d) 6-[1-O-α-(2,3,4,6-Tetra-O-benzyl-D-mannopyranosyl)-hexanoicacid-N-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide

[0195] 100 g (134 mmol) of the acid that is described in Example Ac) and13.5 g (134 mmol) of triethylamine are dissolved in 1,200 ml of drytetrahydrofuran. After cooling to −15° C., a solution of 18.45 g (135mmol) of isobutyl chloroformate in 200 ml of dry tetrahydrofuran isslowly added in drops while being stirred, whereby the internaltemperature does not exceed −10° C. After a reaction time of 15 minutesat −15° C., a solution of 165.5 g (134 mmol) of1-amino-1H,1H,2H,2H-perfluorodecane and 13.5 g (134 mmol) oftriethylamine in 250 ml of dry tetrahydrofuran are added in drops at−20° C. After a reaction time of one hour at −15° C. and two hours atroom temperature, the reaction solution is evaporated to the dry statein a vacuum. The remaining residue is taken up in 300 ml of ethylacetate and washed twice with 400 ml of saturated sodium bicarbonatesolution each and once with 500 ml of water. After the organic phase isdried on sodium sulfate, salt is suctioned out, and the ethyl acetate isremoved in a vacuum. The remaining oily residue is purified on silicagel with use of dichloromethane/hexane/2-propanol (10:5:1) as an eluant.

[0196] Yield: 143.8 g (86.9% of theory)

[0197] Elementary analysis: Cld: C 57.38 H 4.98 N 1.13 F 26.15 Fnd: C57.30 H 5.44 N 1.01 F 26.25

[0198] e) 6-[1-O-α-D-Mannopyranosyl)-hexanoic acidN-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide

[0199] 40.0 g (32.38 mmol) of the title compound of Example Ad) isdissolved in 750 ml of 2-propanol and mixed with 2.0 g of palladiumcatalyst (10% Pd/C). The reaction solution is hydrogenated for 12 hoursat 22° C. and 1 atmosphere of hydrogen pressure.

[0200] Then, catalyst is filtered off, and the filtrate is evaporated tothe dry state. The remaining residue is taken up in 300 ml of dimethylsulfoxide, and 21.52 g (88.0% of theory) of the above-mentioned titlecompound is obtained as a colorless and crystalline powder with thedecomposition melting point of 88.5° C. from the product solution thatis thus obtained by mixing with a total of 1000 ml of diethyl etherafter the precipitated solution is suctioned off.

[0201] Elementary analysis: Cld: C 36.01 H 5.92 N 1.75 F 40.34 Fnd: C36.07 H 6.08 N 1.76 F 40.66

B) Production of the Formulation

[0202] 100 μmol of 6-[1-O-α-D-mannopyranosyl)-hexanoic acidN-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide is dissolvedin 0.5 ml of ethanol, then mixed with 50 ml of water, and 1, 2, 5 and 10mol % of dyes 7 to 14 are added in solid form. After 1 hour of treatmentin an ultrasound bath, dialysis is carried out as described in Example13.

[0203] In addition to 6-[1-O-α-D-mannopyranosyl)-hexanoic acidN-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide, the followingmonosaccharide-perfluoroalkyl derivatives I-III can be used for theproduction of galenical formulations:

I.1-O-α-D-[(1-Perfluoroctylsulfonyl-piperazine-4-carbonyl)-pentyl-5]-mannopyranose

[0204] a)1-O-α-D-[(1-Perfluorooctylsulfonylpiperazine-4-carbonyl)-pentyl-5]-2,3,4,6-tetra-O-benzyl-mannopyranose

[0205] 74.59 g (100 mmol) of the acid that is described in Example Ac)and 10.11 g (100 mmol) of triethylamine are dissolved in 800 ml of amixture of tetrahydrofuran/acetonitrile (mixing ratio 7:3). Then, it ismixed drop by drop at room temperature with 500 ml of a tetrahydrofuransolution of 58.0 g (102.0 mmol) of 1-perfluorooctyl-sulfonylpiperazine;10,11 g (100 mmol) of triethylamine and 16.84 g (110 mmol) of1-hydroxybenzotriazole. The reaction solution that is thus obtained ismixed at −5° C. with a solution of 22.7 g (110 mmol) ofdicyclohexylcarbodiimide, dissolved in 100 ml of tetrahydrofuran, andthen stirred at −5° C. for two more hours. After the reaction solutionhas thawed, it is stirred at room temperature for another 12 hours,precipitated dicyclohexylurea is filtered out, and the filtrate that isobtained is evaporated to the dry state in a vacuum. The remainingresidue is taken up in 600 ml of ethyl acetate and washed twice with 300ml of saturated sodium bicarbonate solution each as well as twice with300 ml of water each.

[0206] After the organic phase is dried on sodium sulfate, salt issuctioned out, and the ethyl acetate is removed in a vacuum. Theremaining oily residue is purified on silica gel with use ofdichloromethane/acetone/2-propanol (16:2:1) as an eluant.

[0207] Yield: 113.01 g (79.8% of theory) of a colorless and viscous oil

[0208] Elementary analysis: Cld: C 58.52 H 4.27 N 1.98 S 2.26 F 22.80Fnd: C 58.42 H 4.41 N 1.80 S 2.28 F 23.02

[0209] b)1-O-α-D-[(1-Perfluorooctylsulfonyl-piperazine-4-carbonyl)-pentyl-5]-mannopyranose

[0210] 50 g (35.30 mmol) of the title compound of Example Ia) isdissolved in a mixture that consists of 500 ml of 2-propanol and 50 mlof water, and 2 g of palladium catalyst (10% Pd on activated carbon) isadded. It is hydrogenated for 12 hours at room temperature. Catalyst isfiltered out, and the filtrate is evaporated to the dry state in avacuum. The residue is dissolved in 200 ml of methanol, and the reactionproduct is precipitated by mixing with a total of 800 ml of diethylether. After the solid that is thus obtained is suctioned off, thelatter is dried in a vacuum at 50° C.

[0211] Yield: 29.51 g (99% of theory) of an amorphous solid

[0212] Elementary analysis: Cld: C 34.13 H 3.46 N 3.32 S 3.80 F 38.23Fnd: C 34.28 H 3.81 N 3.25 S 3.80 F 38.01

II.2-Deoxy-2-[acetyl-(2-amino-N-ethyl-N-perfluorooctylsulfonyl)-amino]-1-α,β-D-mannopyranose

[0213] a)2-Acetamido-2-deoxy-1,3,4,6-(tetra-O-benzyl)-α,β-D-glucopyranose

[0214] A total of 24.0 g (108.5 mmol) of2-acetamido-2-deoxy-α,β-D-glucopyranose, dissolved in 500 ml of absolutedimethyl sulfoxide, is added drop by drop at room temperature to astirred suspension of 20.16 g (700 mmol/80% in mineral oil) of sodiumhydride in 150 ml of dimethyl sulfoxide. Then, it is allowed to stir for120 more minutes at room temperature, and then 159.5 g (1.26 mol) ofbenzyl chloride is added in drops. The reaction solution that is thusobtained is subsequently stirred for another 12 hours at roomtemperature. For working-up, the reaction solution is slowly poured into1.5 liters of ice water and then exhaustively extracted with diethylether. The combined diethyl ether phases are subsequently washed twicewith 600 ml of saturated sodium bicarbonate solution each and twice with800 ml of water each. After the organic phase is dried on sodiumsulfate, salt is suctioned out, and the solvent is removed in a vacuum.The remaining oily residue is purified on silica gel with use of ethylacetate/hexane (1:5) as an eluant.

[0215] Yield: 48.68 g (73.6% of theory) of the above-mentioned titlecompound in the form of a viscous and colorless oil

[0216] Elementary analysis: Cld: C 70.92 H 6.45 N 6.89 Fnd: C 71.43 H6.44 N 7.02

[0217] b)1-O-Benzyl-3,4,6-tri-O-benzyl-2-amino-2-deoxy-α,β-D-glucopyranose

[0218] 30.0 g (49.2 mmol) of the title compound of Example IIa) issuspended in a mixture of 750 ml of methanol and 215 ml of water andmixed drop by drop at room temperature with a total of 440 ml (49.2mmol) of a 0.112 molar aqueous perchloric acid solution. After theaddition is completed, the reaction solution is stirred for 10 moreminutes at room temperature, and the now homogenous reaction solutionthat is thus obtained is subsequently evaporated to the dry state in avacuum. By mixing the remaining oily residue with a mixture thatconsists of equal parts of hexane and dichloromethane, the latter iscrystallized. The crystalline reaction product is suctioned off, washedwith hexane and dried in a vacuum at room temperature.

[0219] Yield: 27.08 g (86% of theory) of the above-mentioned titlecompound in the form of its perchlorate, which is present as acolorless, crystalline compound.

[0220] Melting point: 180.5-181.5° C.

[0221] Elementary analysis: Cld: C 63.68 H 5.98 N 2.19 Cl 5.54 Fnd: C63.43 H 6.04 N 2.02 Cl 5.71

[0222] c)1,3,4,6-Tetra-O-benzyl-2-deoxy-2-[acetyl-(2-amino-N-ethyl-N-perfluorooctylsulfonyl)-amino]-1-α,β-D-mannopyranose

[0223] 20.8 g (35.6 mmol) of the2-[N-ethyl-N-perfluorooctylsulfonyl]-aminoacetic acid and 3.60 g (35.6mmol) of triethylamine are dissolved in 350 ml of dry tetrahydrofuran.After the reaction solution is cooled to −15° C. to −20° C., a solutionof 4.92 g (35.6 mmol) of isobutyl chloroformate in 75 ml of drytetrahydrofuran is slowly added in drops at this temperature while beingstirred, whereby the dropwise addition rate is to be selected so that aninternal temperature of −10° C. is not exceeded. After a reaction timeof 15 minutes at −15° C., a solution of 22.78 g (35.6 mmol) of theperchlorate (title compound of Example IIb) and 3.60 g (35.6 mmol) oftriethylamine, in 100 ml of dry tetrahydrofuran, is then slowly added indrops at −20° C. After a reaction time of one hour at −15° C. and twohours at room temperature, the reaction solution is evaporated to thedry state in a vacuum. The remaining residue is taken up in 250 ml ofethyl acetate and washed twice with 100 ml of saturated sodiumbicarbonate solution each and once with 200 ml of water. After theorganic phase is dried on sodium sulfate, salt is suctioned out, and theethyl acetate is removed in a vacuum. The remaining oily residue ispurified on silica gel with use of ethyl acetate/hexane (1:5) as aneluant.

[0224] Yield: 33.3 g (84.6% of theory) of the above-mentioned titlecompound as a colorless and strongly viscous oil

[0225] Elementary analysis: Cld: C 49.92 H 3.92 N 2.53 F 29.18 S 2.90Fnd: C 49.99 H 4.11 N 2.69 F 29.22 S 3.01

[0226] d)2-Deoxy-2-[acetyl-(2-amino-N-ethyl-N-perfluorooctylsulfonyl)-amino]-1-α,β-D-mannopyranose

[0227] 20.0 g (18.06 mmol) of the title compound of Example IIc) isdissolved in 250 ml of 2-propanol and mixed with 1.5 g of palladiumcatalyst (10% Pd/C). The reaction solution is hydrogenated for 12 hoursat 22° C. and 1 atmosphere of hydrogen pressure. Then, catalyst isfiltered out, and the filtrate is evaporated to the dry state. Theremaining residue is taken up in 300 ml of dimethyl sulfoxide, and 12.65g (93.8% of theory) of the above-mentioned title compound is obtained asa colorless and crystalline powder from the product solution that isthus obtained by mixing with 750 ml of a mixture that consists of equalparts of dimethyl ether and ethyl acetate after the precipitated solidis suctioned off. The above-mentioned title compound is present as anα/β-anomer mixtures, whereby the ratio relative to the two possibleanomers was determined at about 1:1.2 by ¹H-NMR-spectroscopic studies.Accordingly, the title compound is an almost approximately evenlydivided α/β-anomer mixture.

[0228] Melting point: 132.5-133° C.

[0229] Elementary analysis: Cld: C 28.97 H 2.57 N 3.75 F 43.27 S 4.30Fnd: C 29.09 H 2.56 N 3.84 F 43.36 S 4.42

III. 1-O-β-D-[6-Hexanoicacid-N-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide]-glucopyranose

[0230] a) 1,2,3,4,6-Penta-O-acetyl-α-D-glucopyranose

[0231] Analogously, as described in the synthesis of title compound Aa),the reaction of 100 g (555.0 mmol) of α-D-glucopyranose with a mixtureof 1000 ml of absolute pyridine and 1000 ml of acetic acid anhydrideafter working-up and recrystallization from 95% aqueous ethanol yields190.6 g (88.0%) of the above-mentioned title compound as a colorless andcrystalline compound. By ¹H-NMR-spectroscopic study of the titlecompound that is thus obtained, it was possible to determine the α toβ-ratio of two possible anomers with ≧98:2. Accordingly, the titlecompound is the exclusively α-configured anomer.

[0232] Melting point: 110.5° C.

[0233] Elementary analysis: Cld: C 49.21 H 5.68 Fnd: C 49.24 H 5.68

[0234] b)5-(Ethoxycarbonyl)pentyl-2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside

[0235] Analogously, as described in the synthesis of the title compoundof Example Ab), the reaction of 130.0 g (332.8 mmol) of the titlecompound of Example IIIa) with 55.8 ml (332.8 mmol) of6-hydroxy-hexanoic acid ethyl ester and 50.6 ml (520 mmol) of tin(IV)chloride in 500 ml of 1,2-dichloroethane after column-chromatographicworking-up (eluant: hexane/ethyl acetate 2:1) yields 101.85 g (62.4% oftheory) of the above-mentioned title compound as a colorless and viscousoil. After ¹H-NMR-spectroscopic study of the title compound, thepresence of the β-configuration at the anomeric center was definitivelyestablished based on the size of the coupling constant of J_(1,2)=8.8Hz; moreover, said configuration represents the sole existingconfiguration at the anomeric center. It was thus possible to depict theabove-mentioned title compound only in the form of the β-configuredanomer.

[0236] Elementary analysis: Cld: C 52.94 H 6.77 Fnd: C 52.77 H 6.70

[0237] c) 5-(Carboxyypentyl-2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside

[0238] A stirred suspension of 100.0 g (204.96 mmol) of the titlecompound of Example IIIb) in 150 ml of dioxane is mixed at roomtemperature and with simultaneous, vigorous stirring in portions with atotal of 169.14 g (3.02 mol) of finely powdered potassium hydroxidepowder. To increase the stirrability, the reaction mixture is mixed withanother 150 ml of dioxane, and the suspension that is thus obtained issubsequently heated to boiling and mixed drop by drop at thistemperature with a total of 264 ml (2.218 mol) of benzyl bromide over aperiod of two hours. After a reaction time of 4 hours at 110° C.followed by 12 hours at room temperature, the reaction mixture is slowlypoured into a total of 2.0 liters of ice water for the purpose ofworking-up, and the water phase is subsequently completely extractedwith diethyl ether. After the ether phase that is thus obtained iswashed and said phase is subsequently dried on sodium sulfate, salt issuctioned out, and the diethyl ether is removed in a vacuum. Excessbenzyl bromide is then quantitatively distilled off from the reactionmixture in an oil pump vacuum at an oil bath temperature of 180° C. Theremaining oily residue that is thus obtained is purified on silica gelwith use of ethyl acetate/hexane (1:10) as an eluant.

[0239] Yield: 128.8 g (84.3% of theory) of the above-mentioned titlecompound in the form of a colorless and extremely viscous oil

[0240] Elementary analysis: Cld: C 75.68 H 7.16 Fnd: C 75.66 H 7.23

[0241] d) 2,3,4,6-Tetra-O-benzyl-1-O-β-D-[6-hexanoicacid-N-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide]-glucopyranose

[0242] 68.5 g (91.79 mmol) of the acid that is described in ExampleIIIc) and 9.25 g (91.79 mmol) of triethylamine are dissolved in 825 mlof dry tetrahydrofuran. After the reaction solution is cooled to −15° C.to −20° C., a solution of 12.64 g (92.5 mmol) of isobutyl chloroformatein 150 ml of dry tetrahydrofuran is slowly added in drops at thistemperature while being stirred, whereby the dropwise addition rate isto be selected such that an internal temperature of −10° C. is notexceeded. After a reaction time of 15 minutes at −15° C., a solution of46.40 g (91.79 mmol) of1H,1H,2H,2H-heptadecafluoro-1-(2-aminoethyoxy)-decane and 9.25 g (91.79mmol) of triethylamine is then slowly added in drops at −20° C. as asolution in 200 ml of dry tetrahydrofuran. After a reaction time of onehour at −15° C., and two hours at room temperature, the reactionsolution is evaporated to the dry state in a vacuum. The remainingresidue is taken up in 250 ml of ethyl acetate and washed twice with 300ml of saturated sodium bicarbonate solution each and once with 400 ml ofwater. After the organic phase is dried on sodium sulfate, salt issuctioned out, and the ethyl acetate is removed in a vacuum. Theremaining oily residue is purified on silica gel with use ofdichloromethane/hexane/2-propanol (10:5:1) as an eluant.

[0243] Yield: 104.7 g (92.4% of theory) of the above-mentioned titlecompound as a colorless and strongly viscous oil.

[0244] Elementary analysis: Cld: C 57.38 H 4.98 N 1.13 F 26.15 Fnd: C57.27 H 5.09 N 1.11 F 26.08

[0245] e) 1-O-β-D-[6-Hexanoicacid-N-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide]-glucopyranose

[0246] 40.0 g (32.38 mmol) of the title compound of Example IIId) isdissolved in 750 ml of 2-propanol and mixed with 2.0 g of palladiumcatalyst (10% Pd/C). The reaction solution is hydrogenated for 12 hoursat 22° C. and 1 atmosphere of hydrogen pressure. Then, catalyst isfiltered out, and the filtrate is evaporated to the dry state. Theremaining residue is taken up in 300 ml of dimethyl sulfoxide and 22.05g (90.2% of theory) of the title compound is obtained as a colorless andcrystalline powder with a decomposition melting point of 122-124° C.from the product solution that is thus obtained by mixing with a totalof 1000 ml of diethyl ether and subsequent suctioning-off of theprecipitated solid.

[0247] Elementary analysis: Cld: C 36.01 H 5.92 N 1.75 F 40.34 Fnd: C36.07 H 6.08 N 1.76 F 40.66

EXAMPLE 15 Production of galenical formulations of three components inthe example of 6-[1-O-α-D-mannopyranosyl)-hexanoic acidN-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide,10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-Perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecaneand dyes 7 to 14

[0248] The production was performed analogously to the generalinstructions of Example 14 with use of various proportions of6-[1-O-α-D-mannopyranosyl)-hexanoic acidN-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide and10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,and of 5 mol % of dyes 7 to 14 in each case. In this case,6-[1-O-α-D-mannopyranosyl)-hexanoic acidN-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide was dissolvedin ethanol, mixed with a solution of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecanein water, and the mixture was treated in an ultrasound bath after dyewas added, and the procedure was as in Example 13.

EXAMPLE 16 Photophysical Characterization of the Formulations

[0249] The determination of the absorption maxima and extinctioncoefficients was carried out with a Lambda 2-spectrometer (Perkin-ElmerCompany). The fluorescence properties were determined on aSPEX-fluorolog (Instruments S. A. Company, Photomultiplier HamamatsuPM928, excitation 350W xenon lamp). The determination of thefluorescence quantum yield was carried out relative to indocyanine green(O=13% in DMSO) by excitation at 585 nm (dyes with p=2) or 685 nm (dyeswith p=3), in each case of solutions of concentration 2 μM in dye, andcorrection with the spectral sensitivity of lamp and detector.

[0250] Absorption and fluorescence properties of formulations with10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-traazacyclododecanedepending on the dye proportion in the example of dyes 7 and 8Extinction coefficient Fluorescence Mol % of Absorption (L mol⁻¹Fluorescence quantum Dye dye max. (nm) cm⁻¹) max. (nm) yield 7 1 652132,000 681 0.17 7 2 651 120,200 683 0.18 7 5 651 123,700 685 0.12 7 10649 110,900 685 0.04 8 1 755 150,500 784 0.23 8 2 755 148,800 785 0.23 85 753 120,800 788 0.14 8 10 751 108,500 790 0.03

[0251] Absorption and fluorescence properties of the galenicalformulation with10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecaneand dyes 7-14 in a proportion of 5 mol % Extinction coefficientFluorescence Mol % of Absorption (L mol⁻¹ Fluorescence quantum Dye dyemax. (nm) cm⁻¹) max. (nm) yield 7 5 651 123,700 685 0.12 8 5 753 120,800788 0.14 9 5 653 145,000 683 0.14 10 5 754 118,800 788 0.13 11 5 650150,700 684 0.16 12 5 753 124,100 789 0.09 13 5 653 139,400 682 0.12 145 754 120,900 789 0.11

[0252] Aborption and fluorescence properties of the galenicalformulations with 6-[1-O-α-D-mannopyranosyl)-hexanoic acidN-(3-oxo-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide,10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecaneand dye 8 in a proportion of 5 mol % Extinction Mol % Mol % Mol %Absorption coefficient Flourescence Flourescence of of Gd of mono-maximum (L mol⁻¹ maximum quantum Dye Dye complex saccharide (nm) cm⁻¹)(nm) yield 8 5 0 95 760 90500 800 0.16 8 5 10 85 760 102700 800 0.11 8 530 65 760 107900 800 0.14 8 5 50 45 756 108700 798 0.26 8 5 70 25 756111100 794 0.18 8 5 90 5 755 112500 793 0.13

EXAMPLE 17 Production of Lyophilizates and Resuspension

[0253] The galenical formulations that are produced according to theinstructions of Examples 13 to 15 are freeze-dried according to commonprocedures. The blue-colored lyophilizates are stored for 5 days at roomtemperature, then resuspended by shaking in 5-10 ml of water andsubjected to dialysis as described above. The dye content after dialysiswas determined photometrically and in all cases amounted to 96-99%relative to the content before freeze-drying and resuspension.

EXAMPLE 18 Interstitial near-infrared lymphography of guinea pigs with aformulation with 5 mol % of dye 8/95 mol % of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

[0254] The imaging properties of the formulations according to theinvention were studied in vivo in guinea pigs. In this respect, aformulation was administered interstitially/intracutaneously, and theconcentration in regional lymph nodes was observed in a period of 0 to120 minutes. The fluorescence of the substances was stimulated byirradiation of the animals with near-infrared light of wavelength 740nm, which was produced with an Nd:YAG laser. The fluorescence radiationwas detected at a wavelength of >800 nm by an intensified CCD-camera,and the fluorescence images were stored digitally.

[0255]FIG. 1 shows near-infrared fluorescence images of a guinea pigafter interstitial/intracutaneous administration of formulations with 5mol % of dye 8/95 mol % of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane.0.1 ml of a stock solution with 1 mmol/L of 8 was injected into a skinfold between the toes of the right rear extremity, and the finalconcentration was 0.3 μmol/kg of 8.

[0256] A. Abdominal view, 120 minutes after administration, the rightrear foot of the animal was covered during imaging, since the injectionsite had a strong fluorescence signal.

[0257] B. Right lateral view, 117 minutes after administration, theright rear leg of the animal was covered in the imaging, since theinjection site had a strong fluorescence signal.

EXAMPLE 19 Coloring of regional lymph nodes after interstitialadministration of a formulation with 5 mol % of dye 8/95 mol % of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

[0258] After the interstitial near-infrared lymphography was completed,the regional lymph nodes were prepared in the area of the administrationsite. It was observed that these lymph nodes have a green coloringbecause of the accumulation of the substance according to the invention.This coloring can be used for the intraoperative diagnosis for theidentification of the draining lymph nodes of a certain tissue area andfor differentiating the lymph nodes from the surrounding tissue.

[0259]FIG. 2 shows two inguinal lymph nodes of guinea pigs afterinterstitial/intracutaneous administration of a formulation with 5 mol %of dye 8/95 mol % of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane(A, B) and a lymph node of an untreated guinea pig ex vivo (C). Aformulation with 5 mol % of dye 8 resulted in a clearly visiblegreen-coloring of the lymph nodes after interstitial administration.

EXAMPLE 20 Localization of the formulation with 5 mol % of dye 8/95 mol% of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecanein lymph nodes

[0260] The localization of the formulations according to the inventionin lymph nodes was studied by fluorescence microscopy on frozen sectionsof the lymph nodes. In this respect, the lymph nodes were prepared afterthe lymphography and deep-frozen at −80° C. Sections with a 5 μmthickness were produced on a freezing microtome. The evaluation wascarried out on a Zeiss Axiovert 135-fluorescence microscope, which wasequipped with a Cy 7-(excitation filter HQ710/70 nm, emission filter810/90 nm, beam splitter 750 nm LP). From all the preparations, whitelight and fluorescence images were recorded with a CCD camera (PrincetonInstruments RTE/CCD-576) and stored digitally.

[0261]FIG. 3 shows a bright-field (1a) and a near-infrared-fluorescenceimage (1b) of a frozen section of a popliteal guinea pig lymph node fourhours after interstitial administration of a formulation with 5 mol % ofdye 8/95 mol % of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane.Images of a cryosection from a popliteal lymph node of an untreatedguinea pig in bright field (2a) and in near-infrared-fluorescence (2b)are shown for comparison. Lymph nodes from guinea pigs that had receivedan interstitial/intracutaneous injection of the formulation showed apronounced fluorescence in the near-infrared wavelength range, while thelymph nodes of untreated animals have no fluorescence.

[0262] Image parameters:

[0263] Bright field: Lens 2.5×. Exposure 0.02 seconds, no accumulation.Tube enlargement 0.6.

[0264] NTR: Lens 2.5×. Exposure 1 second, accumulation 5×, tubeenlargement 0.6, Cy7-set of filters.

EXAMPLE 21 Fluorescence coloring of lymph nodes after intravenousadministration of a formulation with 5 mol % of dye 8 and 95 mol % of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane.

[0265] From the substance, 0.2 ml of a solution that contained 0.5mmol/L of dye 8 and 9.5 mmol/L of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulflonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,was administered intravenously in a caudal vein of the rat. Five hoursafter administration, the lymph nodes were prepared and a fluorescenceimage was recorded. The fluorescence of the substances was stimulated byirradiation of the lymph nodes with near-infrared light of wavelength740 nm, which was produced with an Nd:YAG laser. The fluorescenceradiation was detected at a wavelength of >800 nm by an intensifiedCCD-camera, and the fluorescence images were stored digitally.

[0266]FIG. 4 shows a fluorescence image of lymph nodes 5 hours afterintravenous administration of a formulation with 5 mol % of dye 8 and 95mol % of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane.Shown are the axillary (a, a′), inguinal (b, b′) and popliteal (c, c′)lymph nodes of the animal. All lymph nodes showed a clear fluorescencesignal.

EXAMPLE 22 Visualization of lymph nodes in situ after interstitialadministration of a formulation with 5 mol % of dye 8 and 95 mol % of6-[1-O-α-D-mannopyranosyl)-hexanoic acidN-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide in rats 0.1 mlof a formulation that contained 1 mmol/L of dye 8 and 19 mmol/L of6-[1-O-α-D-mannopyranosyl)-hexanoic acidN-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide wasadministered interstitially in a skin fold between the toes of the rightrear extremity. Five hours after administration, the test animal wassacrificed and opened abdominally. Based on the fluorescence image, thelymph nodes can be localized in situ and the lymph tracts can bevisualized. The identification of the lymph nodes in situ is alsovisually possible by a green coloring, which is caused afterinterstitial administration of the formulation. The fluorescence of thesubstances was stimulated by irradiation of the abdominally-openedanimal with near-infrared light of wavelength 740 nm, which was producedwith an Nd:YAG laser. The fluorescence radiation was detected at awavelength of >800 nm by an intensified CCD-camera, and the fluorescenceimages were stored digitally.

[0267]FIG. 5 shows a fluorescence image of an abdominally-opened ratfive hours after interstitial administration of a formulation with 5 mol% of dye 8 and 95 mol % of mannose in a skin fold between the toes ofthe right rear extremity. The axillary and the inguinal lymph nodes anda connected lymph tract are visualized based on a fluorescence signal.

EXAMPLE 23 Fluorescence coloring of lymph nodes after interstitialadministration of a formulation with 5 mol % of dye 8 and 95 mol % of6-[1-O-α-D-mannopyranosyl)-hexanoic acidN-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide

[0268] From the substance, 0.1 ml of a solution that contained 1 mmol/Lof dye 8 and 19 mmol/L of 6-[1-O-α-D-mannopyranosyl]-hexanoic acidN-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide wasadministered interstitially in a skin fold between the toes of the rightrear extremity of the animal. Five hours after administration, the lymphnodes of the right and left sides of the body were prepared, and afluorescence image was recorded. The fluorescence of the substances wasstimulated by irradiation of the lymph nodes with near-infrared light ofwavelength 740 nm, which was produced with an Nd:YAG laser. Thefluorescence radiation was detected at a wavelength of >800 nm by anintensified CCD-camera, and the fluorescence images were storeddigitally.

[0269]FIG. 6 shows a fluorescence image of lymph nodes five hours afterinterstitial administration of a formulation with 5 mol % of dye 8 and95 mol % of 6-[1-O-α-D-mannopyranosyl)-hexanoic acidN-(3-oxa-1H,1H,2H,2H,4H,4H,5H,5H-perfluorotridecyl)-amide. Shown are themandibular (m, m′), axillary (a, a′), mesenteric (mes), iliac (il, il′),inguinal (in, in′), gluteal (g, g′) and popliteal (p, p′) lymph nodes ofthe animal. Most lymph nodes of the right (administered) side of thebody showed a fluorescence signal, while no lymph nodes of the left sideof the body (in each case identified with ′) had a fluorescence signal.The fluorescence-labeled lymph nodes were greenish-colored because ofthe accumulation of substances.

[0270] Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. The above preferred specificembodiments are, therefore, to be construed as merely illustrative, andnot limitative of the remainder of the disclosure in any way whatsoever.

[0271] In the foregoing and in the above examples, all temperatures areset forth uncorrected in degrees Celsius; and, unless otherwiseindicated, all parts and percentages are by weight.

[0272] The entire disclosures of all applications, patents andpublications, cited above, and of corresponding German application No.199 48 650.6-43, filed Sep. 29, 1999, and U.S. Provisional ApplicationSerial No. 60/158,306, filed Oct. 8, 1999, are hereby incorporated byreference.

[0273] The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

1. Galenical formulation, characterized in that it containsperfluoroalkyl-containing dye molecules and otherperfluoroalkyl-containing substances.
 2. Formulation according to claim1, wherein the other perfluoroalkyl-containing substances areperfluoroalkyl-containing metal complexes.
 3. Formulation according toclaim 2, wherein the formulations contain otherperfluoroalkyl-containing substances in addition to theperfluoroalkyl-containing dye molecules and theperfluoroalkyl-containing metal complexes.
 4. Formulation according toclaim 1, wherein the perfluoroalkyl-containing dye molecules and theother perfluoroalkyl-containing compounds are present dissolved in asolvent.
 5. Formulation according to claim 4, wherein the solvent iswater.
 6. Formulation according to claim 4, wherein the proportion ofthe perfluoroalkyl-containing dye molecules is between 1 and 10 mol %relative to the total amount of perfluoroalkyl-containing substances. 7.Formulation according to one of claims 1 to 3, wherein theperfluoroalkyl-containing dye molecules absorb and fluoresce in aspectral range of between 400 and 900 nm.
 8. Formulation according toclaim 1, wherein the perfluoroalkyl-containing dye molecules aresubstances of general formula I: R_(f)-L-A  (I) in which R_(f)represents a straight-chain or branched perfluoroalkyl radical with 4 to30 carbon atoms, L stands for a linker, and A stands for a dye molecule.9. Formulation according to claim 8, whereby linker L represents adirect bond or a straight-chain or branched carbon chain with up to 20carbon atoms, which can be substituted with one or more —OH, —COOH, —SO₃groups and/or optionally is interrupted by one or more —O—, —S—, —CO—,—CONH—, —NHCO—, —CONR—, —NRCO—, —SO₂—, —NH—, —NR groups or a piperazine,whereby R stands for a C₁ to C₁₀ alkyl radical, which optionally issubstituted with one or more OH groups and/or is interrupted by one ormore oxygen atoms.
 10. Formulation according to claim 8, wherein dyemolecule A is a dye from the class of polymethine dyes, xanthine dyes orthe heteroaromatic, cationic dyes.
 11. Formulation according to claim 8,wherein dye molecule A is a cyanine dye, squarilium dye, coconium dye,oxonol dye, merocyanine dye, cryptocyanine dye, fluorescein, rhodamine,oxazine, phenoxazine, thiazine or phenothiazine.
 12. Formulationaccording to claim 8, wherein dye molecule A is a molecule according toFormula II:

in which D stands for a fragment that corresponds to general formulasIII to VI, whereby the position that is characterized with a star meansthe linkage with B:

and in which B stands for a fragment that corresponds to generalformulas VII to XII:

whereby R¹ and R² represent a C₁-C₄ sulfoalkyl chain, a saturated orunsaturated, branched or straight-chain C₁-C₅₀ alkyl chain, whereby thechain or parts of this chain optionally can form one or more aromatic orsaturated, cyclic C₅-C₆ units or bicyclic C₁₀ units, and whereby theC₁-C₅₀ alkyl chain optionally is interrupted by 0 to 15 oxygen atomsand/or by 0 to 3 carbonyl groups and/or is substituted with 0 to 5hydroxy groups, R³ stands for a radical —COOE¹, —CONE¹E², —NHCOE¹,—NHCONHE¹, —NE¹E², —OE¹, —OSO₃E¹, —SO₃E¹, —SO₂NHE¹, —E¹, whereby E¹ andE², independently of one another, represent a hydrogen atom, a C₁-C₄sulfoalkyl chain, a saturated or unsaturated, branched or straight-chainC₁-C₅₀ alkyl chain, whereby the chain or parts of this chain optionallycan form one or more aromatic or saturated cyclic C₅-C₆ units orbicyclic C₁₀ units, and whereby the C₁-C₅₀ alkyl chain optionally isinterrupted by 0 to 15 oxygen atoms and/or by 0 to 3 carbonyl groups,and/or is substituted with 0 to 5 hydroxy groups, and whereby R⁴ standsfor a hydrogen atom, a fluorine, chlorine, bromine or iodine atom, or abranched or straight-chain C₁-C₁₀ alkyl chain, b means a number 2 or 3,and X and Y, independently of one another, mean O, S, —CH═CH— orC(CH₃)₂.
 13. Formulation according to claim 2, wherein theperfluoroalkyl-containing metal complexes are selected from thefollowing groups of metal complexes: the gadolinium complex of10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,the gadolinium complex of10-[2-hydroxy-4-aza-5-oxo-7-oxa-10,10,11,11,12,12,13,13,14,14,15,15,16,16,17,17,17-heptadecafluoroheptadecyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,1,4,7-tris{1,4,7-tris(N-carboxylatomethyl)-10-(N-1-methyl-3,6-diaza-2,5,8-trioxooctane-1,8-diyl)-1,4,7,10-tetraazacyclododecane,Gd-complex}-10-(N-2H,2H,4H,4H,5H,5H-3-oxa-perfluoro-tridecanoyl)-1,4,7,10-tetraazacyclododecane,1,4,7-tris{1,4,7-tris[(N-carboxylatomethyl)]-10-[N-1-methyl-3-aza-2,5-dioxopentam-1,5-diyl]-1,4,7,10-tetraazacyclododecane,Gdcomplex}-10-[2-(N-ethyl-N-perfluorooctylsulfonyl)-amino]-acetyl-1,4,7,10-tetraazacyclododecane,the gadolinium complex of10-[2-hydroxy-4-aza-5-oxo-7-aza-7(perfluorooctylsulfonyl)-nonyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,1,4,7-tris(carboxylatomethyl)-10-[(3-aza-4-oxo-hexan-5-ylic)-acid-N-(2,3-dihydroxy-propyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa)-perfluorotridecyl)-amide]-1,4,7,10-tetraazacyclododecane,gadolinium complex,1,4,7-tris(carboxylatomethyl)-10-[(3-aza-4-oxo-hexan-5-ylic)-acid-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa-perfluorotridecyl)-amide]-1,4,7,10-tetraazacyclododecane,gadolinium complex,1,4,7-tris(carboxylatomethyl)-10-{(3-aza-4-oxo-hexan-5-ylic)-acid-[N-3,6,9,12,15-pentaoxa)-hexadexyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa)-perfluorotridecyl]-amide}-1,4,7,10-tetraazacyclododecane,gadolinium complex, and1,4,7-tris(carboxylatomethyl)-10-[(3-aza-4-oxo-hexan-5-ylic)-acid-N-(5-hydroxy-3-oxa-pentyl)-N-(1H,1H,2H,2H,4H,4H,5H,5H-3-oxa)-perfluorotridecyl)-amide]-1,4,7,10-tetraazacyclododecane,gadolinium complex.
 14. Formulation according to claim 3, wherein theadditional perfluoroalkyl-containing substances are compounds of generalformula XV: R_(f)-L²-G¹  (XXXII)in which R_(f) represents astraight-chain or branched perfluoroalkyl radical with 4 to 30 carbonatoms, L² stands for a linker, and G¹ stands for a hydrophilic group.15. Formulation according to claim 14, wherein linker L² is a directbond, an —SO₂ group or a straight-chain or branched carbon chain with upto 20 carbon atoms, which can be substituted with one or more —OH,—COO—, —SO₃ groups and/or optionally contains one or more —O—, —S—,—CO—, —CONH—, —NHCO—, —CONR—, —NRCO—, —SO₂—, —PO₄, —NH—, —NR groups, anaryl ring or a piperazine, whereby R stands for a C₁ to C₂₀ alkylradical, which in turn can contain one or more O-atoms and/or can besubstituted with —COO⁻ or SO₃ groups.
 16. Formulation according to claim14, wherein hydrophilic group G¹ stands for a monosaccharide ordisaccharide, one or more adjacent —COO⁻ or —SO₃ groups, a dicarboxylicacid, an isophthalic acid, a picolinic acid, a benzenesulfonic acid, atetrahydropyrandicarboxylic acid, a 2,6-pyridinedicarboxylic acid, aquaternary ammonium ion, an aminopolycarboxylic acid, anaminodipolyethyleneglycolsulfonic acid, an aminopolyethylene glycolgroup, an SO₂—(CH₂)₂—OH group, a polyhydroxyalkyl chain with at leasttwo hydroxyl groups or one or more polyethylene glycol chains with atleast two glycol units, whereby the polyethylene glycol chains areterminated by an —OH or —OCH₃ group.
 17. Formulation according to claim1, wherein the perfluoroalkyl chains of the perfluoroalkyl-containingdye molecules and the other perfluoroalkyl-containing compounds contain6 to 12 carbon atoms.
 18. Formulation according to claim 17, wherein theperfluoroalkyl chains contain 8 carbon atoms in each case. 19.Substances of general formula I R_(f)-L-A  (I) in which R_(f) representsa straight-chain or branched perfluoroalkyl radical with 4 to 30 carbonatoms, L stands for a linker and A stands for a dye molecule from theclass of polymethine dyes, xanthine dyes or the heteroaromatic, cationicdyes.
 20. Substances according to claim 19, wherein linker L representsa direct bond or a straight-chain or branched carbon chain with up to 20carbon atoms, which can be substituted with one or more —OH, —COOH, —SO₃groups and/or optionally is interrupted by one or more —O—, —S—, —CO—,—CONH—, —NHCO—, —CONR—, —NRCO—, —SO₂—, —NH—, —NR groups or a piperazine,whereby R stands for a C₁ to C₁₀ alkyl radical, which optionally issubstituted with one or more OH groups.
 21. Substances according toclaim 19, wherein dye molecule A is a cyanine dye, squarilium dye,croconium dye, oxonol dye, merocyanine dye, cryptocyanine dye,fluorescein dye, rhodamine dye, oxazine dye, phenoxazine dye, thiazinedye or phenothiazine dye.
 22. Substances according to claim 22, whereindye molecule A is a molecule according to formula II:

in which D stands for a fragment that corresponds to general formulasIII to VI, whereby the position that is characterized with a star meansthe linkage with B:

and in which B stands for a fragment that corresponds to generalformulas VII to XII:

whereby R¹ and R² represent a C₁ -C₄ sulfoalkyl chain, a saturated orunsaturated, branched or straight-chain C₁-C₅₀ alkyl chain, whereby thechain or parts of this chain optionally can form one or more aromatic orsaturated, cyclic C₅-C₆ units or bicyclic C₁₀ units, and whereby theC₁-C₅₀ alkyl chain optionally is interrupted by 0 to 15 oxygen atomsand/or by 0 to 3 carbonyl groups and/or is substituted with 0 to 5hydroxy groups, R³ stands for a radical —COOE¹, —CONE¹E², —NHCOE¹,—NHCONHE¹, —NE¹E², —OE¹, —OSO₃E¹, —SO₃E¹, —SO₂NHE¹, —E¹, whereby E¹ andE², independently of one another, represent a hydrogen atom, a C₁-C₄sulfoalkyl chain, a saturated or unsaturated, branched or straight-chainC₁-C₅₀ alkyl chain, whereby the chain or parts of this chain optionallycan form one or more aromatic or saturated cyclic C₅-C₆ units orbicyclic C₁₀ units, and whereby the C₁-C₅₀ alkyl chain optionally isinterrupted by 0 to 15 oxygen atoms and/or by 0 to 3 carbonyl groups,and/or is substituted with 0 to 5 hydroxy groups, and whereby R⁴ standsfor a hydrogen atom, a fluorine, chlorine, bromine or iodine atom, or abranched or straight-chain C₁-C₁₀ alkyl chain, b means a number 2 or 3,and X and Y, independently of one another, mean O, S, —CH═CH— orC(CH₃)₂.
 23. Process for the production of galenical formulationsaccording to claim 1, wherein the perfluoroalkyl-containing dyemolecules and the other perfluoroalkyl-containing compounds aredissolved in a solvent while being stirred vigorously.
 24. Process forthe production of galenical formulations according to claim 1, whereinthe perfluoroalkyl-containing dye molecules and the otherperfluoroalkyl-containing compounds are dissolved in a solvent whilebeing treated simultaneously with ultrasound.
 25. Process for theproduction of galenical formulations according to claim 1, wherein theperfluoroalkyl-containing dye molecules and the otherperfluoroalkyl-containing compounds are dissolved in a solvent whilebeing treated simultaneously with microwaves.
 26. Process for theproduction of galenical formulations according to claim 1, wherein theperfluoroalkyl-containing dye molecules are dissolved in a solvent, theother perfluoroalkyl-containing compounds are dissolved in anothersolvent, the two solutions are combined, and one of the two solvents isdistilled off.
 27. Solid formulation according to claim 1, wherein it isproduced by freeze-drying a solution, which containsperfluoroalkyl-containing dye molecules and otherperfluoroalkyl-containing substances.
 28. Use of galenical formulationsaccording to claim 1 for the production of contrast media for opticaldiagnosis, fluorescence diagnosis, near-infrared diagnosis, nuclear spintomography, imaging with use of x-rays (x-ray or computer tomography)and for ultrasound imaging.
 29. Use of galenical formulations accordingto claim 1 for the production of contrast media for visualizing lymphnodes or blood pools.
 30. Use of galenical formulations according toclaim 1 for the production of agents for intraoperative diagnosis.